TWI866273B - Nucleotide hemi-sulfate salt for the treatment of hepatitis c virus - Google Patents

Abstract

A hemi-sulfate salt of the structure:

Description

Nucleotide hemisulfate for the treatment of hepatitis C virus

The present invention is hemisulfate salts of selected nucleotide compounds having unexpected therapeutic properties for treating a host infected with hepatitis C, as well as pharmaceutical compositions and dosage forms thereof.

Hepatitis C (HCV) is an RNA single-stranded virus and a member of the Hepacivirus genus. It is estimated that 75% of all cases of liver disease are caused by HCV. HCV infection can lead to cirrhosis and liver cancer, and if left unchecked, can lead to liver failure that may require a liver transplant. Approximately 71 million people worldwide suffer from chronic HCV infection and approximately 399,000 people die from HCV each year, mostly due to cirrhosis and hepatocellular carcinoma. RNA polymerase is a key target for drug development against RNA single-stranded viruses. The HCV nonstructural protein NS5B RNA-dependent RNA polymerase is the key enzyme responsible for initiating and catalyzing viral RNA synthesis. There are two major subclasses of NS5B inhibitors: nucleoside analogs and nonnucleoside inhibitors (NNIs). Nucleoside analogs assimilate into active triphosphates that serve as alternative substrates for the polymerase, and non-nucleoside inhibitors (NNIs) bind to allosteric regions on the protein. Nucleoside or nucleotide inhibitors mimic natural polymerase substrates and act as chain terminators. They inhibit the initiation of RNA transcription and the elongation of nascent RNA chains. In addition to targeting RNA polymerase, other RNA viral proteins can also be targeted in combination therapies. For example, HCV proteins that are additional targets for therapeutic approaches are NS3/4A (serine protease) and NS5A (a nonstructural protein that is an essential component of the HCV replicase and exerts a range of effects on cellular pathways). In December 2013, the first nucleoside NS5B polymerase inhibitor, sofosbuvir (Sovaldi ^® , Gilead Sciences), was approved. Sovaldi ^® is a uridine aminophosphate prodrug that is taken up by hepatocytes and undergoes intracellular activation to yield the active metabolite, 2'-deoxy-2'-α-fluoro-β-C-methyluridine-5'-triphosphate. Sovaldi ^® 2'-Deoxy-2'-α-fluoro-β-C-methyluridine-5'-triphosphate Sovaldi® is the first drug that has shown safety and efficacy in treating certain types of HCV infection without the need for co-administration of interferons. ^Sovaldi® is the third ^drug with breakthrough therapy designation approved by the FDA. In 2014, the U.S. FDA approved ^Harvoni® (ledispasvir, an NS5A inhibitor; and sofosbuvir) for the treatment of chronic hepatitis C virus genotype 1 infection. ^Harvoni® is the first combination pill approved to treat chronic HCV genotype 1 infection. It is also the first approved regimen that does not require administration of interferons or ribavirin. In addition, the FDA approved simeprevir (Olysio ^TM ) in combination with sofosbuvir (Sovaldi ^® ) as a once-daily, all-oral, interferon- and ribavirin-free treatment for adults with genotype 1 HCV infection. The U.S. FDA also approved AbbVie's VIEKIRA Pak ^TM in 2014, a multi-pill package containing dasabuvir (non-nucleoside NS5B polymerase inhibitor), ombitasvir (NS5A inhibitor), paritaprevir (NS3/4A inhibitor) and ritonavir. VIEKIRA Pak ^TM can be used with or without ribavirin to treat patients infected with genotype 1 HCV, including those with compensated cirrhosis. VIEKIRA Pak ^TM does not require co-therapy with interferons. In July 2015, the U.S. FDA approved Technivie ^TM and Daklinza ^TM to treat HCV genotype 4 and HCV genotype 3, respectively. Technivie ^TM (Ombitasvir/Papivir and/ritonavir) is approved for use in combination with ribavirin to treat HCV genotype 4 in patients without scarring and cirrhosis, and is the first choice for patients infected with HCV-4 who do not require co-administration of interferons. Approved Daklinza ^TM for use with Sovaldi ^® to treat HCV genotype 3 infection. Daklinza ^TM is the first drug that has shown safety and efficacy in treating HCV genotype 3 without the need for co-administration of interferons or ribavirin. In October 2015, the U.S. FDA warned that HCV treatments Viekira Pak and Technivie can cause severe liver damage, primarily in patients with underlying advanced liver disease, and requested that additional information about safety be added to the labeling. Other currently approved treatments for HCV include interferon alpha-2b or pegylated interferon alpha-2b (Pegintron ^® ), which can be administered with ribavirin (Rebetol ^® ), NS3/4A telaprevir (Incivek ^® , Vertex and Johnson & Johnson), boceprevir (Victrelis ^™ , Merck), simeprevir (Olysio ^™ , Johnson & Johnson), pamovevir (AbbVie), opitidevir (AbbVie), NNI dasabuvir (ABT-333), and Merck's Zepatier ^™ (a single-tablet combination of two drugs, grazoprevir and elbasvir). Additional NS5B polymerase inhibitors are currently in development. Merck is developing the uridine nucleotide prodrug MK-3682 (formerly Idenix IDX21437) and that drug is currently in Phase II combination trials. U.S. patents and WO applications describing nucleoside polymerase inhibitors for the treatment of flaviviruses, including HCV, include Pharmaceuticals (6,812,219, 6,914,054, 7,105,493, 7,138,376, 7,148,206, 7,157,441, 7,163,929, 7,169,766, 7,192,936, 7,365,057, 7,384,924, 7,4 56,155, 7,547,704, 7,582,618, 7,608,597, 7,608,600, 7,625,875, 7,635,689, 7,662,798, 7,824,851, 7,902,202, 7,932,240, 7,951,789, 8,193,372, 8,299,0 38, 8,343,937, 8,362,068, 8,507,460, 8,637,475, 8,674,085, 8,680,071, 8,69 1,788, 8,742,101, 8,951,985, 9,109,001, 9,243,025, US2016/0002281, US2013 /0064794、WO/2015/095305、WO/2015/081133、WO/2015/061683、WO/2013/17721 9. WO/2013/039920, WO/2014/137930, WO/2014/052638, WO/2012/154321); Merck (6,777,395, 7,105,499, 7,125,855, 7,202,224, 7,323,449, 7,339,054, 7,534,767, 7,632,821, 7,879,815, 8,071,568, 8,148,349, 8,470,834, 8,481,712, 8,541,434, 8,697,694, 8,715,638, 9,061,041, 9,156,872 and WO/2013/009737); Emory University (6,348,587, 6,911,424, 7,307,065, 7,495,006, 7,662,938, 7,772,208, 8,114,994, 8,168,583, 8,609,627, US 2014/0212382 and WO2014/1244430); Gilead Sciences/ Pharmasset Inc. (7,842,672, 7,973,013, 8,008,264, 8,012,941, 8,012,942, 8,318,682, 8,324,179 ,8,415,308, 8,455,451, 8,563,530, 8,841,275, 8,853,171, 8,871,785, 8,877,733 、8,889,159、8,906,880、8,912,321、8,957,045、8,957,046、9,045,520、9,085,573、9,090,642 and 9,139,604) and (6,908,924、6,949,522、7,094,770、7,211,570、7,429,5 72, 7,601,820, 7,638,502, 7,718,790, 7,772,208, RE42,015, 7,919,247, 7,964,58 0, 8,093,380, 8,114,997, 8,173,621, 8,334,270, 8,415,322, 8,481,713, 8,492,53 9, 8,551,973, 8,580,765, 8,618,076, 8,629,263, 8,633,309, 8,642,756, 8,716,262, 8,716,263, 8,735,345, 8,735,372, 8,735,569, 8,759,510 and 8,765,710); Hoffman La-Roche (6,660,721); Roche (6,784,166, 7,608,599, 7,608,601 and 8,071,567); Alios BioPharma Inc. (8,895,723, 8,877,731, 8,871,737, 8,846,896, 8,772,474, 8,980,865, 9,012,427, US 2015/0105341, US 2015/0011497, US 2010/0249068、US2012/0070411、WO 2015/054465、WO 2014/209979、WO 2014/100505、WO 2014/100498, WO 2013/142159, WO 2013/142157, WO 2013/096680, WO 2013/088155, WO 2010/108135); Enanta Pharmaceuticals (US 8,575,119, 8,846,638, 9,085,599, WO 2013/044030, WO 2012/125900); Biota (7,268,119, 7,285,658, 7,713,941, 8,119,607, 8,415,309, 8,501,699 and 8,802,840); Biocryst Pharmaceuticals (7,388,002, 7,429,571, 7,514,410, 7,560,434, 7,994,139, 8,133,870, 8,163,703, 8,242,085 and 8,440,813); Alla Chem, LLC (8,889,701 and WO 2015/053662); Inhibitex (8,759,318 and WO/2012/092484); Janssen Products (8,399,429, 8,431,588, 8,481,510, 8,552,021, 8,933,052, 9,006,29 and 9,012,428); the University of Georgia Foundation (6,348,587, 7,307,065, 7,662,938, 8,168,583, 8,673,926, 8,816,074, 8,921,384 and 8,946,244); RFS Pharma, LLC (8,895,531, 8,859,595, 8,815,829, 8,609,627, 7,560,550, US 2014/0066395, US 2014/0235566, US 2010/0279969, WO/2010/091386 and WO 2012/158811); University College Cardiff Consultants Limited (WO/2014/076490, WO 2010/081082, WO/2008/062206); Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO 2014/169280); Cocrystal Pharma, Inc. (US 9,173,893); Katholieke Universiteit Leuven (WO 2015/158913); Catabasis (WO 2013/090420); and the Regents of the University of Minnesota (WO 2006/004637). Atea Pharmaceuticals, Inc. has disclosed β-D-2'-deoxy-2'-α-fluoro-2'-β-C-substituted-2-modified-N ⁶ -(monomethyl and dimethyl) purine nucleotides for the treatment of HCV in U.S. Patent No. 9,828,410 and PCT Application No. WO 2016/144918. Atea has also disclosed β-D-2'-deoxy-2'-substituted-4'-substituted-2-N ⁶ -substituted-6-amino purine nucleotides for the treatment of paramyxovirus and orthomyxovirus infections in US 2018/0009836 and WO 2018/009623. There remains a strong medical need to develop safe, effective and well-tolerated anti-HCV therapies. This need exacerbates the expectation of drug resistance. More potent direct-acting antivirals can significantly shorten the duration of treatment and improve the compliance and SVR (sustained viral response) rates of patients infected with all HCV genotypes. Therefore, one object of the present invention is to provide compounds, pharmaceutical compositions, methods and dosage forms for treating and/or preventing HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefits of provisional U.S. Application Nos. 62/453,437 filed February 1, 2017, 62/469,912 filed March 10, 2017, 62/488,366 filed April 21, 2017, and 62/575,248 filed October 20, 2017. The entire contents of these applications are incorporated by reference. It has been unexpectedly discovered that the hemisulfate salt of Compound 1 (hereinafter provided as Compound 2 ) exhibits unexpectedly favorable therapeutic properties over its free base (Compound 1 ), including enhanced bioavailability and target organ selectivity. These unexpected advantages could not have been predicted in advance. Therefore, Compound 2 is a therapeutically superior composition of matter for use in treating hepatitis C when administered in an effective amount to a host in need thereof (usually a human). Compound 2 is referred to as (( S )-((( 2R , 3R , 4R , 5R )-5-(2-amino-6-(methylamino) -9H -purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphatyl) -L -alanine isopropyl ester hemisulfate. Compound 1 is disclosed in U.S. Patent No. 9,828,410. Compound 1 Compound 2 Compound 2 (as Compound 1 ) is converted in cells to its corresponding nucleotide triphosphate (Compound 1-6 ), which is an active metabolite and inhibitor of RNA polymerase (see Scheme 1 below). Since Compound 1-6 is produced in cells and does not leave the cells, it is not measurable in plasma. However, the 5'-OH metabolite Compound 1-7 ( see Scheme 1 ) is exported from cells and is therefore measurable in plasma and serves as a surrogate for the concentration of the intracellular active metabolite Compound 1-6 . It has been found that the intravital plasma concentration of the surrogate Compound 1-7 , and therefore the intravital plasma concentration of the intracellular Compound 1-6, is substantially higher when Compound 2 is administered intravitally than when Compound 1 is administered intravitally. In a head-to-head comparison of dogs dosed with Compound 1 and Compound 2 (Example 19, Table 28), administration of Compound 2 achieved an AUC _{( 0-4hrs )} of the final guanine 5'-OH nucleoside metabolite ( 1-7 ) that was _twice the AUC _for Compound 1 administration. Unexpectedly, a non -covalent salt has this effect on the plasma concentration of the parent drug (Compound 1 ). In addition, Compound 2 selectively distributes to the liver in vivo over the heart (Example 19, Table 29), which is beneficial because the liver is a diseased organ in hosts infected with HCV. Dogs were dosed with Compound 1 or Compound 2 and the concentration of active triphosphate (1-6 ) in the liver and heart was measured. As shown in Table 29, the liver to heart ratio of active triphosphate concentrations was higher after dosing Compound 2 compared to Compound 1. Specifically, the liver/heart partitioning ratio of Compound 2 was 20 compared to 3.1 for Compound 1. Unexpectedly, this data indicates that administration of Compound 2 results in a better distribution of active guanine triphosphate (Compounds 1-6 ) in the liver than in the heart compared to Compound 1 , which reduces potential off-target effects. Unexpectedly, administration of Compound 2 significantly reduces undesirable off-target partitioning. This allows, if necessary, the administration of Compound 2 by a healthcare practitioner at a higher dose than Compound 1 . In addition, liver and heart tissue levels of active guanine triphosphate derivatives (metabolites 1-6 ) of Compound 2 were measured in rats and monkeys after oral administration of Compound 2 (Example 20). High levels of active guanine triphosphate ( 1-6 ) were measured in the liver of all species tested. Importantly, incalculable levels of guanine triphosphate ( 1-6 ) were measured in monkey hearts, and this indicates liver-specific formation of the active triphosphate. Thus, it was found that administration of Compound 2 improved the distribution of guanine triphosphate (1-6 ) compared to administration of Compound 1 . When administered to healthy and hepatitis C infected patients, Compound 2 was well tolerated after a single oral dose, and the _Cmax , _Tmax and _AUCtot pharmacokinetic parameters were similar in both groups (Tables 34 and 35). As described in Example 24, a single dose of Compound 2 in HCV infected patients produced significant antiviral activity. Plasma exposure of metabolites 1-7 was mostly proportional to dose within the studied range. Individual pharmacokinetic/pharmacodynamic analysis of patients dosed with Compound 2 showed viral responses that correlated with plasma exposure of metabolites 1-7 of Compound 2 (Example 24, Figures 23A to 23F ) , indicating that deep vial reactions can be achieved with a stable dose of Compound 2 . As a non-limiting example, Example 24 confirms that single oral doses of 300 mg, 400 mg, and 600 mg produce significant antiviral activity in humans. The 600 mg dose of Compound 2 doubled the _C24 minimum plasma concentration of post-metabolites 1-7 of Compound 2 from the 300 mg dose of _Compound 2. Figure 24 and Example 25 highlight the amazing invention provided by Compound 2 for the treatment of hepatitis C. As shown in FIG. 24 , steady-state minimum plasma levels (C _{24 , ss} ) of metabolites 1 - 7 following dosing of Compound 2 (600 mg QD (550 mg free base equivalent) and 450 mg QD (400 mg free base equivalent)) in humans were predicted and compared to the EC ₉₅ of Compound 1 on _a range of HCV clinical isolates in vitro to determine _if steady-state plasma concentrations were consistently above the EC ₉₅ , which would result in higher efficacy against multiple clinical isolates in vivo . The EC ₉₅ of Compound 1 was the same as the EC ₉₅ of Compound 2. For Compound 2 to be effective, the steady-state minimum plasma levels of metabolites 1 - 7 should exceed the EC ₉₅ . As shown in Figure 24, the _EC95 of Compound 2 against all clinical isolates tested ranged from approximately 18 nM to 24 nM. As shown in Figure 24, Compound 2 at a dose of 450 mg QD (400 mg free base equivalent) in humans provided a predicted steady-state minimum plasma concentration of approximately 40 ng/mL ( _{C24 , ss} ). Compound 2 at a dose of 600 mg QD (550 mg free base equivalent) in humans provided a predicted steady-state minimum plasma concentration of approximately 50 ng/mL ( _{C24 , ss} ). Thus, the predicted steady -state plasma concentrations of surrogate metabolites 1-7 were nearly twice the _EC95 for all clinical isolates tested, even the refractory GT3a, indicating excellent potency. In contrast, the _EC95 of the standard of the nucleotide sofosbuvir (Sovaldi) ranged from 50 nM to 265 nM in all HCV clinical isolates tested, with only two isolates, GT2a and GT2b, having _EC95s less than the predicted steady-state concentration at the commercial dose of 400 mg. For the other clinical isolates GT1a, GT1b, GT3a, GT4a, and GT4d, the EC ₉₅ of the commercial dose of 400 mg sofosbuvir was greater than the predicted steady-state concentration. The data comparing the efficacy and pharmacokinetic steady-state parameters in Figure 24 clearly show the unexpected therapeutic importance of Compound 2 for the treatment of hepatitis C. In fact, the predicted steady-state (C _{24 , ss} ) plasma levels after administration of Compound 2 were predicted to be at least 2-fold higher than the EC ₉₅ for all genotypes tested and 3 to 5-fold more potent than GT2. This data indicates that Compound 2 has potent pan-genotypic antiviral activity in humans. As shown in Figure 24, the _EC95 of sofosbuvir against GT1, GT3 and GT4 is greater than 100 ng/mL. Thus, unexpectedly, Compound 2 is active against HCV at a dosage form that delivers a lower steady-state minimum concentration (40-50 ng/mL) than the steady-state minimum concentration achieved by an equivalent sofosbuvir dosage form (approximately 100 ng/mL). Thus, in one embodiment, the present invention includes a dosage form of Compound 2 that provides a steady-state plasma minimum concentration ( _{C24 , ss} ) of metabolites 1-7 between about 15-75 ng/mL, such as 20-60 ng/mL, 25-50 ng/mL, 40-60 ng/mL, or even 40-50 ng/ mL . This is unexpected in view of the fact that the steady-state concentration of the equivalent metabolite of sofosbuvir is about 100 ng/mL. In addition, it has been found that compound 2 is an unusually stable, highly soluble, non-hygroscopic salt with anti-HCV activity. This is unexpected because, with the exception of the hemisulfate salt (compound 2 ), various salts of compound 1 , including the monosulfate salt (compound 3 ), are not physically stable, but instead are deliquescent or become colloidal solids (Example 4), and are therefore not suitable for stable solid pharmaceutical dosage forms. Unexpectedly, although Compound 2 did not become colloidal, its water solubility was up to 43 times higher than Compound 1 , and it was more than 6 times more soluble than Compound 1 under simulated gastric fluid (SGF) conditions (Example 15). As discussed in Example 16, Compound 2 remained as a white solid with an IR corresponding to the 6-month reference standard under accelerated stability conditions (40°C/75%RH). Compound 2 was stable for 9 months under ambient conditions (25°C/60%RH) and freezer conditions (5°C). The solid dosage form of Compound 2 (50 mg and 100 mg tablets) was also chemically stable for 6 months under accelerated (40°C/75%RH) and refrigerated conditions (5°C) (Example 26). Compound 2 is stable as a solid under ambient conditions ( 25°C/60%RH) for at least 9 months. Scheme 1 provides the metabolic pathways of Compound 1 and Compound 2 , which involve the initial deesterification of the aminophosphoric acid ester ( metabolite 1-1 ) to form metabolite 1-2 . Metabolite 1-2 is then converted to ^N6 -methyl-2,6-diaminopurine-5'-monophosphate derivative (metabolite 1-3 ), which in turn is metabolized to free 5'-hydroxy- ^N6 -methyl-2,6-diaminopurine nucleoside (metabolite 1-8 ) and dihydrogen phosphate (( 2R , 3R , 4R , 5R )-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9 - yl ) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran- 2 - yl )methyl ester (metabolite 1-4 ) as 5' - monophosphate. Metabolites 1-4 are assimilated to the corresponding diphosphate (metabolites 1-5 ) and then to the active triphosphate derivative (metabolites 1-6 ). The 5' -triphosphate can be further metabolized to produce 2-amino-9-((2R,3R,4R , 5R ) -3 - fluoro - 4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran- 2 - yl )-1,9-dihydro-6H-purin-6-one (1-7). Metabolites 1-7 are measurable in plasma and are therefore replacements for the active triphosphate (1-6 ) , which is not measurable in plasma. In one embodiment, the present invention is compound 2 and its use for treating hepatitis C (HCV) in a host in need thereof, optionally in a pharmaceutically acceptable carrier. In one aspect, compound 2 is used as an amorphous solid. In another aspect, compound 2 is used as a crystalline solid. The present invention further includes an exemplary non-limiting method for preparing compound 2 , which comprises (i) a first step of dissolving compound 1 in an organic solvent in a flask or container, the organic solvent being, for example, acetone, ethyl acetate, methanol, acetonitrile or diethyl ether; (ii) feeding a second organic solvent into a second flask or container, the second organic solvent being the same or different from the organic solvent in step (i), cooling the second solvent to 0-10° C. as appropriate, and adding H ₂ SO ₄ dropwise to the second organic solvent to produce a H ₂ SO ₄ /organic solvent mixture; wherein the solvent is, for example, methanol; (iii) To the solution of compound 1 in step (i) is added dropwise the H ₂ SO ₄ /solvent mixture in a molar ratio of 0.5/1.0 at ambient temperature or slightly increased or decreased temperature (e.g. 23-35° C.); (iv) the reactants in step (iii) are stirred, for example at ambient temperature or slightly increased or decreased temperature, until a precipitate of compound 2 is formed; (v) the resulting precipitate from step (iv) is filtered as appropriate and washed with an organic solvent; and (vi) the resulting compound 2 is dried under vacuum as appropriate at a higher temperature, for example 55, 56, 57, 58, 59 or 60° C. In one embodiment, the organic solvent in step (i) is 3-methyl-2-pentanone. In one embodiment, the organic solvent in step (i) is ethyl isopropyl ketone. In one embodiment, the organic solvent in step (i) is methyl propionate. In one embodiment, the organic solvent in step (i) is ethyl butyrate. Despite the large number of antiviral nucleoside literature and patent applications, compound 2 has not been specifically disclosed. Therefore, the present invention includes compound 2 or a pharmaceutically acceptable composition or dosage form thereof, as described herein. Compounds, methods, dosage forms and compositions for treating a host infected with HCV virus by administering an effective amount of compound 2 are provided. In certain embodiments, Compound 2 is administered at a dose of at least about 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg. In certain embodiments, Compound 2 is administered for up to 12 weeks, up to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In alternative embodiments, Compound 2 is administered for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks. In certain embodiments, Compound 2 is administered at least once a day or every other day. In certain embodiments, Compound 2 is administered in a dosage form to achieve a steady-state minimum plasma level (C _{24 , ss} ) of metabolites 1 - 7 between about 15-75 ng/mL. In one embodiment, Compound 2 is administered in a dosage form to achieve a steady-state minimum plasma level (C _{24 , ss} ) of metabolites 1 - 7 between about 20-60 ng/mL and. In certain embodiments, Compound 2 is administered in a dosage form to achieve an AUC of metabolites 1 - 7 between about 1,200 ng*h/mL and 3,000 ng*h/mL. In one embodiment, compound 2 is administered in a dosage form to achieve an AUC of metabolites 1-7 between about 1,500 ng*h/mL and 2,100 ng*h/mL. The compounds, compositions and dosage forms may also be used to treat related conditions such as anti-HCV antibody-positive and antigen -positive conditions, viral-based chronic hepatitis, liver cancer (hepatocellular carcinoma (HCC)) caused by advanced hepatitis C, cirrhosis, chronic or acute hepatitis C, fulminant hepatitis C, chronic persistent hepatitis C, and anti-HCV-based fatigue. The compounds or formulations comprising the compounds may also be used prophylactically to prevent or limit the development of clinical disease in individuals who are anti-HCV antibody- or antigen-positive or have been exposed to hepatitis C. Thus, the present invention includes the following features: (a) Compound 2 as described herein; (b) a prodrug of Compound 2 ; (c) use of Compound 2 in the manufacture of a medicament for treating hepatitis C virus infection; (d) use of Compound 2 for treating hepatitis C, optionally in a pharmaceutically acceptable carrier; (e) a method for the manufacture of a medicament intended for the treatment of hepatitis C virus infection, wherein Compound 2 or a pharmaceutically acceptable salt as described herein is used in the manufacture; (f) a pharmaceutical formulation comprising a therapeutically effective amount of Compound 2 and a pharmaceutically acceptable carrier or diluent; (g) a method for preparing a therapeutic product containing an effective amount of Compound 2 ; (h) Solid dosage forms, including those that provide favorable pharmacokinetic profiles; and (i) methods for making Compound 2 , as described herein.

The invention disclosed herein is a compound, method, composition and solid dosage form for treating humans and other host animals infected or exposed to HCV virus, which comprises administering an effective amount of (( S)-((( 2R, 3R, 4R, 5R)-5-(2-amino-6-(methylamino)-9 H-purine-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoyl)- L-Alanine isopropyl hemisulfate (compound 2), which is optionally in a pharmaceutically acceptable carrier. In one embodiment, the compound 2is an amorphous solid. In another embodiment, the compound 2It is a crystalline solid. The compounds, compositions and dosage forms may also be used to treat conditions associated with or resulting from exposure to the HCV virus. For example, the active compounds may be used to treat HCV antibody-positive and HCV antigen-positive conditions, viral-based chronic hepatitis, liver cancer (e.g., hepatocellular carcinoma) caused by advanced hepatitis C, cirrhosis, acute hepatitis C, fulminant hepatitis C, chronic persistent hepatitis C, and anti-HCV-based fatigue. The active compounds and compositions may also be used to treat a range of HCV genotypes. At least six different genotypes of HCV have been identified worldwide, each of which has multiple subtypes. Genotypes 1-3 are prevalent worldwide, while genotypes 4, 5, and 6 are more geographically restricted. Genotype 4 is common in the Middle East and Africa. Genotype 5 is mostly found in South Africa. Genotype 6 is found primarily in Southeast Asia. Although the most common genotype in the United States is genotype 1, defining the genotype and subtype can help with the type and duration of treatment. For example, different genotypes respond differently to different drugs, and the optimal duration of treatment varies depending on the genotype infected. Within a genotype, subtypes (such as genotype 1a and genotype 1b) also respond differently to treatment. Infection with one type of genotype does not preclude a later infection with a different genotype. As described in Example 22, compound 2Active against a range of HCV genotypes (including genotypes 1-5). In one embodiment, the compound 2Used to treat HCV genotype 1, HCV genotype 2, HCV genotype 3, HCV genotype 4, HCV genotype 5 or HVC genotype 6. In one embodiment, the compound 2For the treatment of HCV genotype 1a. In one embodiment, the compound 2For the treatment of HCV genotype 1b. In one embodiment, the compound 2For the treatment of HCV genotype 2a. In one embodiment, the compound 2For the treatment of HCV genotype 2b. In one embodiment, the compound 2For the treatment of HCV genotype 3a. In one embodiment, the compound 2For the treatment of HCV genotype 4a. In one embodiment, the compound 2For the treatment of HCV genotype 4d. In one embodiment, the compound 1or compound 2For the treatment of HCV genotype 5a. In one embodiment, the compound 1or compound 2For the treatment of HCV genotype 6a. In one embodiment, the compound 1or compound 2For the treatment of HCV genotype 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n, 6o, 6p, 6q, 6r, 6s, 6t or 6u. As discussed in Example 25 and shown in Figure 24, a dose of 450 mg (400 mg free base) and a dose of 600 mg (550 mg free base) of the compound 2Descendants of the past1 - 7The predicted minimum steady-state concentration (C _{twenty four , ss}) is about 40 ng/mL to 50 ng/mL. This C _{twenty four , ss}Levels exceeded those of compounds in HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, and 4d 1EC ₉₅. This data confirms that the compound 2Possesses potent pan-genotypic activity. This is surprising because the compound 2After administration of an equivalent dose of sofosbuvir, the minimum steady-state concentration of nucleoside metabolites (C_{twenty four , ss}) Smaller stable minimum concentration (C _{twenty four , ss}). The minimum stable concentration of the corresponding nucleoside metabolite of sofosbuvir (C _{twenty four , ss}) is about 100 ng/mL, but this level just exceeds the EC of sofosbuvir for the GT2 clinical isolate ₉₅(Figure 24). Compounds 2A dosage form that is more potent than sofosbuvir against GT1, GT2, GT3, and GT4, and therefore allows for delivery of a smaller steady-state minimum concentration of its metabolite that is still effective against all tested genotypes of HCV. In one embodiment, delivery achieves metabolites between approximately 15-75 ng/mL 1 - 7Steady-state minimum concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between approximately 20-60 ng/mL, 20-50 ng/mL, or 20-40 ng/mL.1 - 7Steady-state minimum concentration (C _{twenty four , ss}) compounds 2In one embodiment, the compound, formulation, or solid dosage form comprising the compound may also be used prophylactically to prevent or delay the development of clinical symptoms in individuals who are HCV antibody or HCV antigen positive or have been exposed to hepatitis C. In particular, it has been found that compared to its free base (compound 1), compound 2It is active against HCV and exhibits excellent drug-like and pharmacological properties. Unexpectedly, the compound 2is more bioavailable and effective than compounds 1Higher AUC (Example 19), and compound 2Compounds 1More selective for target organs (Example 19). In terms of solubility and chemical stability, the compound 2Also better than compounds 1. This is unexpected because (( S)-((( 2R, 3R, 4R, 5R)-5-(2-amino-6-(methylamino)-9 H-purine-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoyl)- L-Alanine isopropyl ester (compound 3) is unstable and presents a viscous gel-like form, while the compound 2(Hemisulfate salt) is a stable white solid. The hemisulfate salt (both as solid and in solid dosage form) is extremely stable and non-hygroscopic over 9 months. Despite the large literature and patent applications for antiviral nucleosides, no specific compounds have yet been revealed 2. Compounds 2It has S stereochemistry at the phosphorus atom, which has been confirmed by X-ray crystallography (Figure 3, Example 2). In an alternative embodiment, the compound 2The phosphorus R and S enantiomers may be used in any desired ratio (including to the extent of enantiomer purity). In some embodiments, the compound 2Used in a form that is at least 90% free of the opposite enantiomer and may be at least 98%, 99% or even 100% free of the opposite enantiomer. Unless otherwise stated, enantiomerically enriched compounds 2At least 90% free of the opposite enantiomer. In addition, in an alternative embodiment, the amino acid of the aminophosphoric acid ester may be in the D-configuration or the L-configuration or a mixture thereof, including a racemic mixture. Unless otherwise specified, the compounds described herein are provided in the β-D-configuration. In an alternative embodiment, the compound may be provided in the β-L-configuration. Similarly, any substituent that exhibits chirality may be provided in racemic, enantiomeric, diastereomeric form or any mixture thereof. When the aminophosphoric acid ester exhibits chirality, it may be provided as an R or S chiral phosphorus derivative or a mixture thereof, including a racemic mixture. All such combinations of stereo configurations are alternative embodiments of the invention described herein. In another embodiment, the compound 2At least one of the hydrogen atoms in (nucleotide or hemisulfate) may be replaced by deuterium. Such substitution configurations include (but are not limited to), I. (( S )-((( 2R , 3R , 4R , 5R )- 5 -( 2 - Amine - 6 -( Methylamino )- 9 H - Purine - 9 - base )- 4 - fluorine - 3 - Hydroxyl - 4 - Methyltetrahydrofuran - 2 - base ) Methoxy )( Phenoxy ) Phosphate )- L - Alanine isopropyl hemisulfate ( Compound 2 )The active compound of the present invention is a compound 2, which can be provided in a pharmaceutically acceptable composition or solid dosage form. In one embodiment, the compound 2is an amorphous solid. In yet another embodiment, the compound 2It is a crystalline solid. Compound 2 SynthesisThe present invention further includes a non-limiting illustrative method for preparing compound 2, comprising: (i) in a flask or container, placing compound1The first step is to dissolve in an organic solvent, such as acetone, ethyl acetate, methanol, acetonitrile or ether; (ii)    A second organic solvent is added into a second flask or container, which may be the same or different from the organic solvent in step (i), and the second solvent is cooled to 0-10°C as appropriate, and H is added dropwise to the second organic solvent.₂SO ₄, producing H ₂SO ₄/ organic solvent mixture; wherein the solvent may be methanol, for example; (iii)   at ambient temperature or a slightly increased or decreased temperature (e.g. 23-35°C), to the compound of step (i) 1Add H from step (ii) dropwise to the solution at a molar ratio of 0.5/1.0.₂SO ₄/solvent mixture; (iv)   stirring the reactants of step (iii) at, for example, ambient temperature or at a slightly increased or decreased temperature until a compound is formed 2 (v)    optionally filtering the precipitate obtained from step (iv) and washing with an organic solvent; and (vi)   optionally drying the obtained compound under vacuum at a higher temperature, such as 55, 56, 57, 58, 59 or 60°C 2. In certain embodiments, the above step (i) is carried out in acetone. In addition, the second organic solvent in step (ii) may be, for example, methanol, and the mixture of organic solvents in step (v) is methanol/acetone. In one embodiment, in step (i), the compound 1Dissolved in ethyl acetate. In one embodiment, in step (i), the compound 1Dissolved in tetrahydrofuran. In one embodiment, in step (i), the compound 1Dissolved in acetonitrile. In one embodiment, in step (i), the compound 1Dissolved in dimethylformamide. In one embodiment, the second organic solvent in step (ii) is ethanol. In one embodiment, the second organic solvent in step (ii) is isopropanol. In one embodiment, the second organic solvent in step (ii) is n-butanol. In one embodiment, a mixture of solvents is used for washing in step (v), such as ethanol/acetone. In one embodiment, the mixture of solvents used for washing in step (v) is isopropanol/acetone. In one embodiment, the mixture of solvents used for washing in step (v) is n-butanol/acetone. In one embodiment, the mixture of solvents used for washing in step (v) is ethanol/ethyl acetate. In one embodiment, the mixture of solvents used for washing in step (v) is isopropanol/ethyl acetate. In one embodiment, the mixture of solvents used for washing in step (v) is n-butanol/ethyl acetate. In one embodiment, the mixture of solvents used for washing in step (v) is ethanol/tetrahydrofuran. In one embodiment, the mixture of solvents used for washing in step (v) is isopropanol/tetrahydrofuran. In one embodiment, the mixture of solvents used for washing in step (v) is n-butanol/tetrahydrofuran. In one embodiment, the mixture of solvents used for washing in step (v) is ethanol/acetonitrile. In one embodiment, the mixture of solvents used for washing in step (v) is isopropanol/acetonitrile. In one embodiment, the mixture of solvents used for washing in step (v) is n-butanol/acetonitrile. In one embodiment, the mixture of solvents used for washing in step (v) is ethanol/dimethylformamide. In one embodiment, the mixture of solvents used for washing in step (v) is isopropanol/dimethylformamide. In one embodiment, the mixture of solvents used for washing in step (v) is n-butanol/dimethylformamide. II. (( S )-((( 2R , 3R , 4R , 5R )- 5 -( 2 - Amine - 6 -( Methylamino )- 9 H - Purine - 9 - base )- 4 - fluorine - 3 - Hydroxyl - 4 - Methyltetrahydrofuran - 2 - base ) Methoxy )( Phenoxy ) Phosphate )- L - Isopropyl alanine ( Compound 2 ) MetabolismCompounds 1and compounds 2Metabolism involves the generation of 5'-monophosphate and subsequent N ⁶-Methyl-2,6-diaminopurine ( 1 - 3) is assimilated to produce dihydrogen phosphate (( 2R, 3R, 4R, 5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purine-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl ester ( 1 - 4). The monophosphate is then further assimilated into the active triphosphate species, 5'-triphosphate ( 1 - 6). The 5'-triphosphate can be further metabolized to produce 2-amino-9-(( 2R, 3R, 4R, 5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one ( 1 - 7). Alternatively, the 5'-monophosphate 1-2 can be metabolized to produce the purine base 1 - 8. (( S)-((( 2R, 3R, 4R, 5R)-5-(2-amino-6-(methylamino)-9 H-purine-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoyl)- LThe metabolic pathway of isopropyl-alanine is illustrated in Scheme 1 (shown above). III. Compound 1 Additional saltIn an alternative embodiment, the present invention provides an oxalate salt (compound 4) or in the form of HCl salt (compound 5) compounds 1. Both the 1:1 oxalate salt and the 1:1 HCl salt form solids with reasonable properties for a solid dosage form for use in treating a host, such as a human with hepatitis C. However, less oxalate salt may be required, and it may not be appropriate if the patient is susceptible to kidney stones. The HCl salt is more hygroscopic than the hemisulfate salt. Therefore, the hemisulfate salt remains the most desirable salt form of Compound 1 with unexpected properties. IV. DefinitionAs used in the context of the present invention, the term "D-configuration" refers to a configuration in principle that mimics the natural configuration of the sugar moiety as opposed to the non-naturally occurring nucleoside or "L" configuration. The term "β" or "β rotamer" is used with reference to nucleoside analogs in which the nucleoside base is configured (placed) on the plane of the furanose moiety in the nucleoside analog. The term "coadminister" or combination therapy is used to describe the administration of a compound according to the present invention in combination with at least one other active agent, such as, if appropriate, at least one additional anti-HCV agent 2. The timing of co-administration is best determined by the medical professional treating the patient. Sometimes it is better to administer the various agents simultaneously. Alternatively, the drugs selected for combination therapy can be administered to the patient at different times. Of course, when more than one viral or other infection or other condition is present, the compounds of the invention can be combined with other agents as needed to treat the other infection or condition. As used herein, the term "host" refers to a single-cell or multicellular organism in which the HCV virus can replicate, including cell strains and animals, and generally humans. The term host specifically refers to infected cells, cells and animals transfected with all or part of the HCV genome, specifically primates (including chimpanzees) and humans. In most animal applications of the invention, the host is a human patient. However, in certain indications, the invention clearly contemplates applications in veterinary medicine (such as chimpanzees). The host may be, for example, a cow, horse, bird, dog, cat, etc. Isotope substitutionThe present invention includes compounds and compounds having isotopic substitutions of desired atoms 2, the amount of the isotope substituted is above the natural abundance of the isotope, i.e., enriched. Isotopes are atoms with the same atomic number but different mass numbers (i.e., the same number of protons but different numbers of neutrons). By way of general example and not limitation, hydrogen isotopes, such as deuterium ( ²H) and tritium ( ³H). Alternatively or additionally, carbon isotopes may be used, such as ¹³C and ¹⁴C. Preferred isotopic substitutions are substitutions of deuterium for hydrogen at one or more positions on the molecule to improve the potency of the drug. Deuterium can be incorporated at positions that cleave bonds during metabolism (α-deuterium kinetic isotope effect) or that are proximal to or near the bond cleavage site (β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describe deuteration of nucleotides to improve their pharmacokinetic or pharmacodynamic properties, including at the 5-position of the molecule. Substitution with isotopes such as deuterium may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. The substitution of deuterium for hydrogen at a metabolic decomposition site can reduce the rate of metabolism at that bond or eliminate metabolism at that bond. At any position in a compound where a hydrogen atom may exist, the hydrogen atom may be any isotope of hydrogen, including protium ( ¹H), deuterium ( ²H) and tritium ( ³H). Therefore, unless the context clearly dictates otherwise, references to compounds herein encompass all possible isotopic forms. The term "isotopically labeled" analogs refers to "deuterated analogs", "¹³C-labeled analogues" or "deuterated/ ¹³C-labeled analogs". The term "deuterated analogs" means compounds described herein in which the H-isotope (i.e., hydrogen/protium ( ¹H)) through the H-isotope (that is, deuterium ( ²H)) substitution. Deuterium substitution can be partial or complete. Partial deuterium substitution means that at least one hydrogen is replaced by at least one deuterium. In certain embodiments, the isotope is enriched by 90%, 95%, or 99% or more for any isotope at any position of interest. In some embodiments, the 90%, 95%, or 99% enrichment at the desired position is deuterium. Unless otherwise indicated, the deuteration is at least 80% at the selected position. Deuteration of the nucleoside can occur at any replaceable hydrogen that provides the desired result. V. Treatment or prevention methodsAs used herein, treatment refers to administering a compound to a host (e.g., a human who may become infected with the HCV virus).2. The term "prophylactic" or "preventive" when used refers to the administration of a compound 2To prevent the occurrence of the viral disease or reduce the possibility of the occurrence of the viral disease. The present invention includes both therapeutic and prophylactic or preventive treatments. In one embodiment, the compound is administered to a host that has been exposed to hepatitis C virus infection and is therefore at risk of contracting hepatitis C virus infection.2. The present invention relates to a method for treating or preventing: Hepatitis C virus, including drug-resistant and multi-drug-resistant forms of HCV and related disease states, conditions; or complications of HCV infection, including cirrhosis and related liver poisoning; and other conditions secondary to HCV infection, such as weakness, loss of appetite, weight loss, breast enlargement (especially in men), rash (especially on the palms), blood clotting problems, spider veins on the skin, confusion, coma (encephalopathy), accumulation of fluid in the abdominal cavity (ascites), esophageal varices, portal hypertension, renal failure, enlarged spleen, low blood cells, anemia, thrombocytopenia, jaundice and hepatocellular carcinoma, and other diseases. The method comprises administering an effective amount of a compound as described herein to a host (usually a human) in need thereof.2, which is optionally combined with at least one additional biologically active agent (e.g., an additional anti-HCV agent), and in addition with a pharmaceutically acceptable carrier, additive and/or formulation. In yet another embodiment, the present invention is a method for preventing or controlling: HCV infection or disease condition or related or subsequent disease condition, HCV infection condition or complication, including cirrhosis and related liver poisoning, weakness, loss of appetite, weight loss, breast enlargement (especially in men), rash (especially on the palms), blood clotting problems, spider veins on the skin, confusion, coma (encephalopathy ), accumulation of fluid in the abdominal cavity (ascites), esophageal varices, portal hypertension, renal failure, splenomegaly, low blood counts, anemia, thrombocytopenia, jaundice and hepatocellular (liver) cancer, as well as other diseases, the method comprising administering to a patient at risk an effective amount of a compound as described above in combination with a pharmaceutically acceptable carrier, additive or excipient (optionally in combination with another anti-HCV agent) 2. In another embodiment, the active compound of the present invention may be administered to a patient after transplantation of a hepatitis-related liver to protect the new organ. In an alternative embodiment, the compound 2Provided as compounds 1Hemisulfate salts of aminophosphoesters other than the specific aminophosphoesters described in the compound description. A wide range of aminophosphoesters are known to those skilled in the art and include various esters and phosphates, in any combination, that can be used to provide the active compound as described herein in the form of a hemisulfate salt. VI. Pharmaceutical compositions and dosage formsIn one embodiment of the present invention, the pharmaceutical composition according to the present invention comprises an anti-HCV virus effective amount of the compound as described herein 2, which is optionally combined with a pharmaceutically acceptable carrier, additive or excipient, and optionally combined or alternating with at least one other active compound. In one embodiment, the present invention includes a compound in a pharmaceutically acceptable carrier 2Solid dosage form. In one embodiment of the present invention, the pharmaceutical composition according to the present invention comprises an anti-HCV effective amount of the compound described herein 2, which is combined with a pharmaceutically acceptable carrier, additive or excipient as appropriate, and is also combined with at least one other antiviral agent (such as an anti-HCV agent) as appropriate. The present invention includes a pharmaceutical composition comprising an effective amount of a compound of the present invention for treating hepatitis C virus infection in a pharmaceutically acceptable carrier or excipient 2Or prodrug. In an alternative embodiment, the present invention includes a pharmaceutical composition comprising an effective amount of the compound of the present invention for preventing hepatitis C virus infection in a pharmaceutically acceptable carrier or formulation 2or prodrugs. Those skilled in the art will recognize that the therapeutically effective amount will vary depending on the infection or condition to be treated, its severity, the treatment regimen to be adopted, the pharmacokinetics of the agent used, and the patient or individual (animal or human) to be treated, and such therapeutic amount can be determined by the attending physician or specialist. Compounds according to the present invention 2Can be formulated in admixture with a pharmaceutically acceptable carrier. In general, it is preferred to administer the pharmaceutical composition in an orally administrable form (and in particular, a solid dosage form such as a pill or tablet). Certain formulations may be administered parenterally, intravenously, intramuscularly, topically, transdermally, intrabuccally, subcutaneously, by suppository or other routes (including intranasal spray). Intravenous and intramuscular formulations are usually administered in sterile saline form. One of ordinary skill in the art may modify the formulation to render it more soluble in water or another vehicle, for example, this may be readily accomplished by minor modifications (salt formulation, esterification, etc.) that are well within the ordinary skill of the art. Modification of Compounds 2It is also well within the skill of the routine practitioner to control the pharmacokinetics of the compounds of the invention for maximum beneficial effect in patients, as described in more detail herein. In certain pharmaceutical dosage forms, prodrug forms of the compounds, including, inter alia, acylated (acetylated or otherwise), and ether (alkyl and related) derivatives, phosphates, thioamidophosphorylates, phosphamidons and various salt forms of the compounds of the invention may be used to achieve the desired effect. One of ordinary skill in the art will recognize how to readily modify the compounds of the invention into prodrug form to facilitate delivery of the active compound to a target site within a host organism or patient. One skilled in the art will also, where applicable, utilize the favorable pharmacokinetic parameters of the prodrug form in delivering the compounds of the invention to target sites within a host organism or patient to maximize the intended effect of the compound. Compounds included in therapeutically active formulations according to the invention 2The amount is an effective amount to achieve the desired result according to the present invention, for example, for treating HCV infection, reducing the likelihood of HCV infection, or inhibiting, reducing and/or eliminating HCV or its secondary effects, including disease states, conditions and/or complications secondary to the occurrence of HCV. Generally speaking, the therapeutically effective amount of the compounds of the present invention in the form of pharmaceutical dosage forms can range from about 0.001 mg/kg to about 100 mg/kg or more per day, more usually ranging from slightly less than about 0.1 mg/kg to more than about 25 mg/kg of the patient per day or significantly higher, depending on the compound used, the condition or infection treated, and the route of administration. Compounds 2The dosage is usually administered in an amount ranging from about 0.1 mg/kg to about 15 mg/kg per patient per day, depending on the pharmacokinetics of the agent in the patient. This dosage range generally produces effective blood level concentrations of the active compound, which can range from about 0.001 to about 100, about 0.05 to about 100 micrograms per cubic centimeter of blood in the patient. Usually, in order to treat, prevent or delay the onset of such infections and/or to reduce the likelihood of HCV viral infection or secondary disease conditions, conditions or complications of HCV, the compound 2The solid dosage form should be administered at least once a day in an amount ranging from about 250 micrograms up to about 800 milligrams or more, for example, once, twice, three times, or up to four times a day in an amount of at least about 5, 10, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 milligrams or more, as directed by a healthcare provider. Compounds 2Administration is usually orally, but may be parenterally, topically or in the form of a suppository, and intranasally (as a nasal spray or in another form described herein). More generally, the compound 2It can be administered in the form of tablets, capsules, injections, intravenous formulations, suspensions, liquids, emulsions, implants, granules, spheres, creams, ointments, suppositories, inhalable forms, transdermal forms, intrabuccal, sublingual, topical, gel, transmucosal and the like. Unless otherwise specified, when the dosage form herein refers to a milligram weight dose, it refers to the compound 2(i.e., the weight of the hemisulfate salt). In certain embodiments, the pharmaceutical composition contains the following amount of compound in a unit dosage form 2Dosage form: about 1 mg to about 2000 mg, about 10 mg to about 1000 mg, about 100 mg to about 800 mg, about 200 mg to about 600 mg, about 300 mg to about 500 mg, or about 400 mg to about 450 mg. In certain embodiments, the pharmaceutical composition is in a dosage form, for example, in a unit dosage form containing the following amount of compound 2Solid dosage forms of: about 10 mg, about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, or about 1000 mg or more. In one embodiment, the compound 2Administration is in a dosage form that delivers at least about 300 mg. In one embodiment, the compound 2The compound is administered in a dosage form that delivers at least about 400 mg. In one embodiment, the compound 2The compound is administered in a dosage form that delivers at least about 500 mg. In one embodiment, the compound 2Administration is in a dosage form that delivers at least about 600 mg. In one embodiment, the compound 2The compound is administered in a dosage form that delivers at least about 700 mg. In one embodiment, the compound 2Administered in a dosage form that delivers at least about 800 mg. In certain embodiments, the compound 2Administered at least once a day for up to 12 weeks. In certain embodiments, the compound 2Administered at least once a day for up to 10 weeks. In certain embodiments, the compound 2Administered at least once a day for up to 8 weeks. In certain embodiments, the compound 2Administered at least once a day for up to 6 weeks. In certain embodiments, the compound 2Administered at least once a day for up to 4 weeks. In certain embodiments, the compound 2Administered at least once a day for at least 4 weeks. In certain embodiments, the compound 2Administered at least once a day for at least 6 weeks. In certain embodiments, the compound 2Administered at least once a day for at least 8 weeks. In certain embodiments, the compound 2Administered at least once a day for at least 10 weeks. In certain embodiments, the compound 2Administered at least once a day for at least 12 weeks. In certain embodiments, the compound 2Administered at least every other day for up to 12 weeks, up to 10 weeks, up to 8 weeks, up to 6 weeks, or up to 4 weeks. In certain embodiments, the compound 2The compound is administered at least every other day for at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks, or at least 12 weeks. In one embodiment, at least about 600 mg of the compound is administered at least once a day.2Up to 6 weeks. In one embodiment, at least about 500 mg of the compound is administered at least once a day 2Up to 6 weeks. In one embodiment, at least about 400 mg of the compound is administered at least once a day 2Up to 6 weeks. In one embodiment, at least 300 mg of the compound is administered at least once a day 2Up to 6 weeks. In one embodiment, at least 200 mg of the compound is administered at least once a day 2Up to 6 weeks. In one embodiment, at least 100 mg of the compound is administered at least once a day 2Up to 6 weeks. Metabolites 1 - 6For active compounds 2triphosphate, but metabolites 1 - 6Not measurable in plasma. Metabolites 1 - 6Substituted as metabolites 1 - 7. Metabolites 1 - 7It is a nucleoside metabolite that is measurable in plasma and is therefore a metabolite 1 - 6Indication of intracellular concentration. For maximum HCV antiviral activity, compound 2The dosage form must achieve more than the compound 2EC ₉₅Metabolites of value 1 - 7Steady-state minimum concentration (C _{twenty four , ss}). As shown in Figure 24, compounds targeting clinical isolates of GT1, GT2, GT3, and GT4 1EC ₉₅Less than 25 ng/mL (compound 1EC ₉₅and compounds 2EC ₉₅value). In one embodiment, delivery results in a metabolite between approximately 15 and 75 ng/mL 1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 60 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 30 and 60 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 50 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 30 and 50 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 45 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 30 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 35 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2In one embodiment, the delivery achieves a metabolite between about 20 and 25 ng/mL.1 - 7The minimum stable concentration (C _{twenty four , ss}) compounds 2The dosage form is ±10% of the minimum stable concentration. In one embodiment, the compound 2To achieve metabolites between approximately 1,200 and 3,000 ng/mL 1 - 7AUC (area under the curve) is administered. In one embodiment, the compound 2To achieve metabolites between approximately 1,500 and 3,000 ng/mL 1 - 7AUC is administered. In one embodiment, the compound 2To achieve metabolites between approximately 1,800 and 3,000 ng/mL 1 - 7AUC is administered. In one embodiment, the compound 2With metabolites between approximately 2,100 and 3,000 ng/mL 1 - 7AUC is administered. In a preferred embodiment, the compound 2To achieve a metabolite of approximately 2,200 ng*h/mL 1 - 7AUC is administered. Suitable dosage forms are ±10% of AUC. In combination with another anti-HCV compound as described herein 2In the case of, the compound according to the present invention to be administered 2The amount of the second active agent is between about 0.01 mg/kg of the patient to about 800 mg/kg or more of the patient or significantly higher, depending on the second agent to be co-administered and its efficacy against the virus, the patient's condition and the severity of the disease or infection to be treated, and the route of administration. Other anti-HCV agents can be administered, for example, in an amount ranging from about 0.01 mg/kg to about 800 mg/kg. An example of a dosage of the second active agent is an amount ranging from about 250 micrograms to about 750 mg or more, for example, at least about 5, 10, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700 or 800 milligrams or more, at least once a day up to four times a day. In certain preferred embodiments, the compound 2The dosage may generally be administered in an amount ranging from about 0.5 mg/kg to about 50 mg/kg or more (usually up to about 100 mg/kg), which generally depends on the pharmacokinetics of both agents in the patient. Such dosage ranges generally produce effective blood level concentrations of the active compound in the patient. For purposes of the present invention, a prophylactic or preventive effective amount of a composition according to the present invention falls within the same concentration range as described above for a therapeutically effective amount, and is generally the same as a therapeutically effective amount. Compounds 2Administration may range from continuous (intravenous infusion) to several oral or intranasal administrations (e.g., Q.I.D.) or transdermal administrations per day, and may include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), intrabuccal, and suppository administrations, among other routes of administration. Oral tablets coated with enteric coatings may also be used to enhance the bioavailability of the compound for oral routes of administration. The most effective dosage form will depend on the bioavailability/pharmacokinetics of the particular agent selected and the severity of the patient's disease. Oral dosage forms are particularly preferred because of their ease of administration and expected favorable patient compliance. To prepare the pharmaceutical composition according to the present invention, a therapeutically effective amount of the compound according to the present invention is usually added according to the known pharmaceutical compounding techniques for producing dosages.2Intimately mixed with a pharmaceutically acceptable carrier. The carrier can take a wide variety of forms depending on the desired formulation for administration (e.g., oral or parenteral). When preparing oral dosage forms of pharmaceutical compositions, any of the common pharmaceutical media can be used. Thus, for liquid oral formulations (such as suspensions, elixirs and solutions), suitable carriers and additives can be used, including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives may be used, including starches, sugar carriers such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrants and the like. If necessary, tablets or capsules may be enteric coated or sustained release by standard techniques. The use of such dosage forms can significantly enhance the bioavailability of the compound in the patient's body. For parenteral formulations, the carrier will generally comprise sterile water or aqueous sodium chloride solution, but may also include other ingredients, including those that aid dispersion. Of course, in the case of using sterile water and maintaining sterility, the composition and carrier must also be sterilized. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents and the like can be used. Liposomal suspensions (including liposomes targeting viral antigens) can also be prepared by known methods to produce pharmaceutically acceptable carriers. This can be suitable for delivering free nucleosides, acyl/alkyl nucleosides or phosphate prodrug forms of nucleoside compounds according to the present invention. In typical embodiments according to the present invention, the compounds described 2and compositions for treating, preventing or delaying HCV infection or secondary disease conditions, symptoms or complications of HCV. VII. Combination and Alternation TherapyIt is well recognized that drug-resistant variants of the virus may emerge after long-term treatment with antiviral agents. Drug resistance sometimes develops through mutations in genes encoding enzymes used in viral replication. The effect of a drug against HCV infection may be prolonged, potentiated, or restored by combining or alternating the administration of the compound with another, and possibly even two or three, other antiviral compounds that induce different mutations or act via different pathways than the primary drug. Alternatively, such combination therapy (which may include alternation therapy if considered synergistic) may be utilized to alter the pharmacokinetics, biodistribution, half-life, or other parameters of the drug. Due to the disclosed compounds 2The compound is an NS5B polymerase inhibitor, so it can be suitable for administration to a host in combination with, for example, the following: (1)    a protease inhibitor, such as an NS3/4A protease inhibitor; (2)    an NS5A inhibitor; (3)    another NS5B polymerase inhibitor; (4)    an NS5B non-substrate inhibitor; (5)    interferon alpha-2a, which can be pegylated or otherwise modified, and/or ribavirin; (6)    a non-substrate inhibitor; (7)    a helicase inhibitor; (8)    an antisense oligodeoxynucleotide (S-ODN); (9)    an aptamer; (10)  a nuclease-resistant ribonuclease; (11)  iRNA, including microRNA and siRNA; (12) Antibodies, partial antibodies or domain antibodies of viruses, or (13) Viral antigens or partial antigens that induce host antibody responses. Can be used with the compounds of the present invention 2Non-limiting examples of anti-HCV agents for combination administration (either alone or with multiple drugs from this list) are: (i)     Protease inhibitors, such as Telaprevir (Incivek ^®）、Poprevi (Victrelis ^TM）、Simipivi (Olysio ^TM), ABT-450, glecaprevir (ABT-493), Norvir, ACH-2684, AZD-7295, BMS-791325, danoprevir, Filibuvir, GS-9256, GS-9451, MK-5172, Setrobuvir, Sovaprevir, Tegobuvir, VX-135, VX-222 and ALS-220; (ii) NS5A inhibitors, such as ACH-2928, ACH-3102, IDX-719, daclatasvir, ledipasvir, velpatasvir (Epclusa), elbavir (MK-8742), grazopivir (MK-5172) and opititevir (ABT-267); (iii)   NS5B inhibitors, such as AZD-7295, Clemizole, Exviera, ITX-5061, PPI-461, PPI-688, sofosbuvir (Sovaldi)^®), MK-3682 and mericitabine; (iv)   NS5B inhibitors, such as ABT-333 and MBX-700; (v)   Antibodies, such as GS-6624; (vi)   Combination drugs, such as Harvoni (ledipasvir/sofosbuvir), Viekira Pak (obitasvir/papivir/ritonavir/dasabuvir), Vikla (obitasvir/papivir/ritonavir), G/P (papivir and gcapivir), Technivie (obitasvir/papivir/ritonavir) and Epclusa (sofosbuvir/velpatasvir) and Zepatier (elbavir and gezopivir). In one embodiment, if the compound is administered 2To treat advanced hepatitis C virus causing liver cancer or cirrhosis, the compound can be administered in combination or alternating with another drug commonly used to treat hepatocellular carcinoma (HCC), such as described by Andrew Zhu in "New Agents on the Horizon in Hepatocellular Carcinoma" Therapeutic Advances in Medical Oncology, V 5(1), January 2013, 41-50. In the case where the host has HCC or is at risk for HCC, examples of suitable compounds for combination therapy include anti-angiogenic agents, sunitinib, brivanib, linifanib, ramucirumab, bevacizumab, cediranib, pazopanib, TSU-68, lenvatinib, anti-EGFR antibodies, mTor inhibitors, MEK inhibitors, and histone deacetylase inhibitors. Examples General MethodsRecorded on a 400 MHz Fourier transform Brücker spectrometer ¹H. ¹⁹F and ³¹P NMR spectra. Unless otherwise stated, DMSO-d ₆Spectra. Rotational multimodality is indicated by the symbols s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad peak). Coupling constants are reported in Hz ( J). Reactions were generally performed under dry nitrogen atmosphere using Sigma-Aldrich anhydrous solvents. All common chemicals were purchased from commercial sources. The following abbreviations are used in the examples: AUC: Area Under the Curve C _{twenty four}: 24-hour drug concentration in plasma C _{twenty four , ss}: Concentration 24 hours after administration under steady state C _max: Maximum concentration of drug achieved in plasma DCM: dichloromethane EtOAc: ethyl acetate EtOH: ethanol HPLC: high pressure liquid chromatography NaOH: sodium hydroxide Na ₂SO ₄：Sodium sulfate (anhydrous) MeCN：Acetonitrile MeNH ₂: methylamine MeOH: methanol Na ₂SO ₄：Sodium sulfate NaHCO ₃：Sodium bicarbonate NH ₄Cl: ammonium chloride NH ₄OH: ammonium hydroxide PE: petroleum ether Ph ₃P: triphenylphosphine RH: relative humidity silica gel (230 to 400 mesh, adsorbent) t-BuMgCl: tert-butylmagnesium chloride T _max：Achieve C _maxtime THF: tetrahydrofuran (THF), anhydrous TP: triphosphate Examples 1 . Compound 1 Synthesis Steps 1 : ( 2R , 3R , 4R , 5R )- 5 -( 2 - Amine - 6 -( Methylamino )- 9 H - Purine - 9 - base )- 4 - fluorine - 2 -( Hydroxymethyl )- 4 - Methyltetrahydrofuran - 3 - alcohol ( 2 - 2 ) SynthesisMethanol (30 L) was added to a 50 L flask and stirred at 10 ± 5°C. NH ₂CH ₃(3.95 Kg) was slowly aerated into the reactor. Compounds were added in batches at 20 ± 5°C.2- 1(3.77 kg) and stirred for 1 hour to obtain a clear solution. The reaction was stirred for an additional 6 to 8 hours, at which time HPLC indicated that the intermediate was less than 0.1% of the solution. Solid NaOH (254 g) was fed to the reactor, stirred for 30 minutes, and concentrated at 50 ± 5°C (vacuum level: -0.095). EtOH (40 L) was fed to the resulting residue and reslurried at 60°C for 1 hour. The mixture was then filtered through diatomaceous earth and the filter cake was reslurried with EtOH (15 L) at 60°C for 1 hour. The filtrate was filtered again, combined with the filtrate from the previous filtration, and then concentrated at 50 ± 5°C (vacuum level: -0.095). A large amount of solid precipitated. EtOAc (6 L) was added to the solid residue, and the mixture was concentrated at 50 ± 5°C (vacuum level: -0.095). Then, DCM was added to the residue, and the mixture was re-slurried at reflux for 1 hour, cooled to room temperature, filtered, and dried in a vacuum oven at 50 ± 5°C to obtain the compound as an off-white solid 2- 2(1.89 Kg, 95.3%, purity 99.2%). Compound 2 - 2 Of Analytical methods :The compounds were obtained using an Agilent 1100 HPLC system with an Agilent Poroshell 120 EC-C18 4.6 × 150 mm 4 μm column under the following conditions 2- 2Purity of (15 mg): 1 mL/min flow rate, read at 254 nm, 30°C column temperature, 15 μL injection volume, and 31 min run time. The sample was dissolved in acetonitrile-water (20:80) (v/v). The gradient method is shown below. Time (min ) A% (0.05 TFA / water ) B% ( acetonitrile ) 0 95 5 8 80 20 13 50 50 twenty three 5 95 26 5 95 26.1 95 5 31 95 5 Steps 2 : (( S )-((( 2R , 3R , 4R , 5R )- 5 -( 2 - Amine - 6 -( Methylamino )- 9 H - Purine - 9 - base )- 4 - fluorine - 3 - Hydroxyl - 4 - Methyltetrahydrofuran - 2 - base ) Methoxy )( Phenoxy ) Phosphate )- L - Isopropyl alanine ( Compound 1 ) SynthesisCompound2- 2and compounds 2- 3(((Perfluorophenoxy)(phenoxy)phosphoyl)- L -Alanine isopropyl ester) was dissolved in THF (1 L) and stirred under nitrogen. The suspension was then cooled to below -5°C and 1.7 M t -BuMgCl solution (384 mL). Add NH to the suspension at room temperature.₄Cl (2 L) and water (8 L) solution, followed by DCM. After adding 5% K ₂CO ₃The mixture was stirred for 5 min before being added to an aqueous solution (10 L) and for an additional 5 min before being filtered through celite (500 g). The celite was washed with DCM and the filtrate was separated. The organic phase was purified by 5% K ₂CO ₃Aqueous solution (10 L × 2), saline solution (10 L × 3), and Na ₂SO ₄(500 g) and dried for about 1 hour. At the same time, this entire process was repeated 7 times in parallel, and the 8 batches were combined. The organic phase was filtered and concentrated at 45 ± 5°C (vacuum level 0.09 Mpa). EtOAc was added, and the mixture was stirred at 60°C for 1 hour, and then stirred at room temperature for 18 hours. Then, the mixture was filtered and washed with EtOAc (2 L) to obtain the crude compound 1. The crude material was dissolved in DCM (12 L), heptane (18 L) was added at 10-20°C, and the mixture was stirred at this temperature for 30 minutes. The mixture was filtered, washed with heptane (5 L), and dried at 50 ± 5°C to obtain the pure compound 1(1650 g, 60%). Compound 1 Analysis Method :The compounds were obtained using an Agilent 1100 HPLC system with a Waters XTerra Phenyl 5 μm 4.6 × 250 mm column under the following conditions 1Purity of (25 mg): 1 mL/min flow rate, read at 254 nm, 30°C column temperature, 15 μL injection volume, and 25 min run time. The sample was dissolved in acetonitrile-water (50:50) (v/v). The gradient method is shown below. Time (min) A% (0.1% H ₃ PO ₄ in water ) B% ( acetonitrile ) 0 90 10 20 20 80 20.1 90 10 25 90 10 Examples 2 . Amorphous and crystalline compounds 1 FeaturesInitially through XRPD, ¹HNMR and HPLC analysis of amorphous compounds 1and crystalline compounds 1. The XRPD patterns of the two compounds are shown in Figure 1A, and the HPLC traces for purity determination are shown in Figure 1B and Figure 2A, respectively. Table 1 is from the crystalline compounds 1Table 2 is a list of XRPDs, and Table 3 is a list of relative retention times (RTTs) from HPLC traces. Amorphous compounds 1is 98.61% pure, and crystalline compound 1 is 99.11% pure. Both compounds are white solids. Figure 2B is the crystalline compound 1TGA and DSC graphs. For crystalline compounds 1For , an endotherm was observed at 88.6°C, and there was a 7.8% mass loss at 80-110°C. Compound 1The sample was recrystallized from EtOAc/hexane and extracted using ORTEP. The compound was confirmed by recrystallized single crystals 1The absolute structure of the compound. Figure 3 shows the compound 1ORTEP diagram. The crystallization data and measurement data are shown in Table 3. Compounds based on X-ray crystallization 1The absolute stereochemistry is shown below: . DSC data were collected on a TA Instruments Q2000 equipped with a 50-position autosampler. Calibration of heat capacity was performed using sapphire, and calibration of energy and temperature was performed using certified indium. Approximately 3 mg of each sample was heated from 25°C to 200°C in a pin-hole aluminum pan at typically 10°C/min. A dry nitrogen purge was maintained over the sample at 50 mL/min. The instrument control software was Advantage for the Q series v2.8.0.394 and Thermal Advantage v5.5.3, and data were analyzed using Universal Analysis v4.5A. TGA data were collected on a TA Instruments Q500 TGA equipped with a 16-position autosampler. The instrument was temperature calibrated using certified Alumel and Nickel. Typically 5 to 10 mg of each sample was loaded onto a front tared aluminum DSC pan and heated from ambient to 350°C at 10°C/min. A nitrogen purge was maintained over the sample at 60 mL/min. The instrument control software was Q Series Advantage v2.5.0.256 and Thermal Advantage v5.5.3, and Universal Analysis v4.5 was used to analyze the data. Amorphous compounds 1 ( 1 - 1 ) : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 1.01 - 1.15 (m, 9 H), 1.21 (d, J=7.20 Hz, 3 H), 2.75 3.08 (m, 3 H), 3.71 - 3.87 (m, 1 H), 4.02 - 4.13 (m, 1 H), 4.22 - 4.53 (m, 3 H), 4.81 (s , 1 H), 5.69 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.44 (m, 3 H), 7.81 (s, 1 H) Crystalline compounds 1 ( 1 - 2 ) : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.97 - 1.16 (m, 16 H), 1.21 (d, J=7.07 Hz, 3 H), 2.87 (br s, 3 H), 3.08 (s, 2 H), 3.79 (br d, J=7.07 Hz, 1 H), 4.08 (br d, J=7.58 Hz, 1 H), 4.17 - 4.55 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.78 (br s, 1 H), 5.91 - 6.15 (m, 4 H), 7.10 - 7.26 (m, 3 H), 7.26 - 7.44 (m, 3 H), 7.81 (s , 1 H) surface 1 . Crystalline compounds 1 Peak List Angle /°2θ d spacing/Å Intensity / Count Strength /% 6.03 14.64 1005 39.0 7.36 12.00 315 12.2 7.94 11.13 1724 66.9 9.34 9.47 2500 97.0 9.51 9.29 860 33.4 9.77 9.05 1591 61.8 11.08 7.98 2576 100.0 12.02 7.36 171 6.6 12.95 6.83 319 12.4 13.98 6.33 241 9.4 14.30 6.19 550 21.4 14.69 6.03 328 12.7 15.20 5.82 2176 84.5 15.94 5.56 1446 56.1 16.75 5.29 1009 39.2 17.29 5.13 700 27.2 17.72 5.00 1213 47.1 18.11 4.89 1565 60.8 18.46 4.80 302 11.7 18.89 4.69 385 14.9 19.63 4.52 636 24.7 20.37 4.36 1214 47.1 20.74 4.28 1198 46.5 21.24 4.18 640 24.8 22.31 3.98 961 37.3 22.88 3.88 806 31.3 23.43 3.79 355 13.8 24.08 3.69 573 22.2 24.49 3.63 159 6.2 25.00 3.56 351 13.6 25.36 3.51 293 11.4 26.09 3.41 235 9.1 26.26 3.39 301 11.7 26.83 3.32 696 27.0 27.35 3.26 436 16.9 27.46 3.25 363 14.1 28.07 3.18 200 7.8 28.30 3.15 195 7.6 28.82 3.10 599 23.3 29.85 2.99 217 8.4 30.26 2.95 186 7.2 30.75 2.91 333 12.9 31.12 2.87 149 5.8 31.85 2.81 238 9.2 33.28 2.69 261 10.1 34.77 2.58 171 6.6 35.18 2.55 175 6.8 36.83 2.44 327 12.7 37.41 2.40 172 6.7 surface 2.From amorphous compounds 1and crystalline compounds 1Relative retention time of HPLC analysis Amorphous Compound 1 Crystalline compound 1 RRT Area% RRT Area% 0.48 0.15 0.48 0.17 0.51 0.04 0.48 0.17 0.48 0.15 0.94 0.12 0.51 0.04 1.00 99.11 0.94 0.13 1.04 0.22 0.98 0.21 1.37 0.07 1.00 98.61 1.04 0.29 1.37 0.31 surface 3 . Compound 1 Crystallization and data measurement Key accuracy CC = 0.0297A, wavelength = 1.54184 Cells a=10.1884(3) b=28.6482(9) c=12.9497(5) α=90 β=113.184(4) γ=90 temperature 150K Calculated value Report value Volume 3474.5(2) 3474.5(2) Space Group P21 P 1 21 1 Hall Group P2 P2 Partial C24 H34 F N7 O7 P 2(C24 H34 F N7 O7 P) Total C24 H34 F N7 O7 P C48 H68 F2 N14 O14 P2 Mr 582.55 1165.10 Dx, g cm ^-1 1.114 1.114 Z 4 2 Mu (mm ^-1 ) 1.139 1.139 F000 1228.0 1228.0 F000' 1233.21 h, k, l _max 12,34,15 12,34,15 N _ref 12742 [ 6510] 8259 _Tmin , _Tmax 0.790, 0.815 0.808, 1.000 T _{min '} 0.716 Calibration method #Reported T Limits: T _min = 0.808 T _max = 1.00 AbsCorr MULTI-SCAN Data Integrity 1.27/0.65 θ (max) 68.244 R (Reflection) 0.2091 ( 7995) wR2 (Reflection) 0.5338 ( 8259) S 2.875 Npar 716 After this initial characterization, the samples were stored at 25°C/60% relative humidity (RH) for 14 days, and HPLC and XRPD analysis was performed after 7 and 14 days. Figure 4A shows the XRPD after 14 days at 25°C/60% (RH). Amorphous compound 1(Sample 1-1) remained poorly crystallized, although the crystallized compound 1(Sample 1-2) retains its crystallinity, but both compounds are stable after 14 days at 25°C/60% (RH). Examples 3 . Oxalate compounds 4 FormationInitially, the compound was formed by mixing oxalate with a solvent (5 vol, 100 μL)1Oxalate compounds 4Any solution was allowed to vaporize at room temperature. Any suspension was allowed to cure (room temperature to 50°C) for 3 hours and the degree of crystallinity was obtained. Table 4 shows the generated compounds 4The different solvents used in the reaction. Except for two (cyclohexane and n-heptane), all solvents gave crystalline products. Although the compound 4The higher crystallinity and solubility of oxalate, but oxalate is not available for clinical research due to the potential formation of kidney stones and compounds.1Other salts. surface 4 . Oxalate compounds 4 Formation Solvent Observation after adding acid at room temperature Observations after aging / evaporation EtOH Solution OXA - Form 1 IPA Solution OXA - Form 1 acetone Solution OXA - Form 1 MEK Solution OXA - Form 1 EtOAc Suspension OXA - Form 1 i Pr Suspension OXA - Form 1 THF Solution OXA - Form 1 Toluene Solution OXA - Form 1 MeCN Solution OXA - Form 1 IPA: 10% water Solution OXA - Form 1 TBME Suspension OXA - Form 1 Cyclohexane Suspension Amorphous n-Heptane Suspension Amorphous Examples 4 . Amorphous compounds 1 Salt compoundsDue to oxalate compounds 4(Example 3) It may not be used in clinical trials due to its potential to form kidney stones, so the anti-ion forming compounds listed in Table 5 1The amorphous salt of. The compound 1Dissolved in tert-butanol (20 vol, 6 ml), and the solution treated with acid counterion (1 eq for each sample except sample 1-9 (which had 0.5 eq of sulfate)). The samples were then frozen, and in this case the solvent was removed by freeze drying. The residual solids in samples 1-4, 1-5, 1-6, 1-7, 1-8, and 1-9 were initially analyzed by XRPD and HPLC. surface 5 . Amorphous salt formation details Sample ID Sample Details Stock solution details Observation NMR 1-4 HCl (1:1) THF 1M White solid 3 less protons ~ 0.3 eq t -BuOH 1-5 Sulfuric acid (1:1) THF 1M White solid 3 less protons ~ 0.3 eq t -BuOH 1-6 Fumaric acid (1:1) MeOH:THF (1:1) 0.5M Glassy solid 1.05 eq fumaric acid 0.84 eq t -BuOH 1-7 Benzoic acid (1:1) THF 1M White solid 1.0 eq benzoic acid 0.34 eq t -BuOH 1-8 Succinic acid (1:1) MeOH 1M Viscous white solid ~ 1.1 eq succinic acid 0.37 eq t -BuOH 1-9 Sulfuric acid (0.5:1 acid:API) THF 1M White solid 3 less protons ~ 0.3 eq t -BuOH All samples were subjected to ¹HNMR spectroscopy analysis. Sample 1 - 4 , HCl ( 1 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.93 - 1.39 (m, 16 H), 2.97 (br s, 2 H), 3.70 - 3.88 (m, 1 H), 4.10 (br s, 1 H), 4.18 - 4.49 (m, 3 H) , 4.70 - 4.88 (m, 1 H), 5.71 - 5.94 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H), 7.14 - 7.27 (m, 3 H), 7.29 - 7.44 (m, 2 H), 7.83 - 8.19 (m, 1 H) Sample 1 - 5 , sulfuric acid ( 1 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.97 - 1.38 (m, 15 H), 2.96 (br s, 2 H), 4.06 - 4.18 (m, 1 H), 4.19 - 4.49 (m, 3 H), 4.66 - 4.91 (m, 1 H ), 5.70 - 5.95 (m, 1 H), 5.96 - 6.16 (m, 2 H), 7.10 - 7.27 (m, 3 H), 7.30 - 7.43 (m, 2 H), 7.88 - 8.19 (m, 1 H) Sample 1 - 6 , Fumaric acid ( 1 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.95 - 1.31 (m, 21 H), 2.87 (br s, 3 H), 3.79 (br d, J=7.20 Hz, 1 H), 4.01 - 4.13 (m, 1 H), 4.16 - 4.23 (m, 1 H), 4.16 - 4.24 (m, 1 H), 4.20 (s, 1 H), 4.18 - 4.23 ( m, 1 H), 4.24 - 4.52 (m, 1 H), 4.24 - 4.52 (m, 1 H), 4.24 - 4.49 (m, 1 H), 4.72 - 4.88 (m, 1 H), 5.68 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 6.63 (s, 1 H), 6.61 - 6.66 (m, 1 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.45 (m, 3 H), 7.81 (s, 1 H), 13.16 (br s, 2 H) Sample 1 - 7 , benzoic acid ( 1 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.96 - 1.30 (m, 15 H), 2.87 (br s, 3 H), 3.79 (br d, J=7.07 Hz, 1 H), 4.07 (br s, 1 H), 4.20 (s, 1 H), 4.25 - 4.52 (m, 3 H), 4.81 (s, 1 H), 5.71 - 5.85 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.08 - 7.27 (m, 3 H), 7.27 - 7.43 (m, 3 H), 7.45 - 7.57 (m, 2 H), 7.63 (s, 1 H), 7.81 (s, 1 H), 7.95 (dd, J=8.27, 1.33 Hz, 2 H), 12.98 (br s, 1 H) Sample 1 - 8 , Succinic acid ( 1 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.98 - 1.28 (m, 15 H), 2.42 (s, 5 H), 2.87 (br s, 3 H), 3.57 - 3.62 (m, 1 H), 3.70 - 3.86 (m, 1 H), 4.02 - 4.14 (m, 1H), 4.20 (s, 1 H), 4.24 - 4.51 (m, 3 H), 4.70 - 4.88 (m, 1 H), 5.69 - 5.86 (m, 1 H), 6.04 (br d, J=19.33 Hz, 4 H), 7.12 - 7.27 (m, 3 H), 7.27 - 7.44 (m, 3 H), 7.81 (s, 1 H), 11.95 - 12.58 (m, 2 H) Sample 1 - 9 , sulfuric acid ( 0 . 5 : 1 ) salt : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 1.02 - 1.31 (m, 15 H), 2.94 (br s, 3 H), 3.79 (br d, J=7.20 Hz, 2 H), 4.09 (br s, 1 H), 4.22 - 4.48 (m, 3 H), 4.72 - 4.90 (m, 1 H), 5.71 - 5.92 (m, 1 H), 6.07 (br d, J=19.07 Hz, 2 H), 7.12 - 7.28 (m, 3 H), 7.31 - 7.44 (m, 2 H), 7.75 - 8.19 (m, 1 H). The samples were then stored at 25°C/60% relative humidity (RH) for 14 days, with HPLC and XRPD analysis performed after 7 days ( FIG. 4B ) and 14 days ( FIG. 5A ). All prepared salts remained amorphous and the observations are shown in Table 6. The monosulfate salts (samples 1-5) and the succinate salts (samples 1-8) were found to be physically unstable and deliquesced or became gelatinous during the study period. Both the fumarate salt (sample 1-6) and the benzoate salt (sample 1-7) were found to be glassy solids. The HCl salt (sample 1-4) was found to retain its physical form. Surprisingly, the hemisulfate salt (sample 1-9) also retained its physical form as a white solid, compared to the monosulfate compound (sample 1-5) which was a viscous gel. The results are shown in Table 6. The mono-HCl salt (sample 1-4) and the hemisulfate salt (sample 1-9) were found to be physically and chemically stable after storage at 25°C/60% relative humidity (RH) for 2 weeks. Although both salts were stable over two weeks, the hemisulfate salt was preferred over the HCl salt because the HCl salt is hygroscopic, rendering it less suitable for long-term storage or use than the hemisulfate salt. surface 6 . Sample in 25 ℃ / 60 % RH Down 7 Tianji 14 The stability of the Queen Sample ID Exposure time at 25℃/60% RH ( days ) 0 7 14 HPLC Observation HPLC Observation HPLC Observation 1-1 98.6 White solid 98.7 White solid 98.5 White solid 1-2 99.1 White solid 99.2 White solid 99.0 White solid 1-3 99.7 White solid 99.6 White solid 99.4 White solid 1-4 98.7 White solid 98.8 White solid 98.6 White solid 1-5 98.4 White solid 55.7 Viscous white solid - Viscous gel 1-6 98.7 Glassy solid 98.6 Transparent glassy solid 98.4 White glassy solid 1-7 98.8 White solid 98.8 Transparent glassy solid 98.7 Transparent glassy solid 1-8 98.7 Viscous white solid - Deliquescent/viscous oily - deliquescence 1-9 98.7 White solid 98.1 White solid 96.4 White solid Examples 5 . Amorphous compounds 2 FeaturesInitially through XRPD, ¹Analysis of amorphous compounds by HNMR, DSC, TGA and HPLC 2. Amorphous compounds 2With amorphous compounds 1and crystalline compounds 1The superimposed XRPD pattern is shown in Figure 1A, and the amorphous compound alone 2The XRPD pattern of is shown in FIG5B. Table 7 is a list of peaks from the XRPD pattern shown in FIG5B. The HPLC trace for purity determination is shown in FIG6A. Table 8 is a list of relative retention times (RTT) from the HPLC trace shown in FIG6A. Amorphous Compounds 299.68% pure. Figure 6B is an amorphous compound 2TGA and DSC graphs. The experimental details of TGA and DSC experiments are given in Example 2. surface 7.Amorphous compounds 2Peak List Angle/°2θ d spacing/Å Intensity / Count Strength/% 4.20 21.03 486 81.8 4.67 18.91 482 81.0 5.16 17.10 595 100.0 9.13 9.68 547 92.0 surface8. HPLC chromatogram of amorphous compound 2 Amorphous Compound 2 RRT Area % 0.48 0.02 0.48 0.02 0.67 0.01 0.94 0.13 1.00 99.68 1.04 0.06 Amorphous compounds 2 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.93 - 1.29 (m, 13 H), 2.94 (br s, 3 H), 3.79 (td, J=10.04, 7.07 Hz, 2 H), 4.05 - 4.19 (m, 1 H), 4.19 - 4.50 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.71 - 5.94 (m, 1 H), 5.97 - 6.16 (m, 2 H), 7.14 - 7.28 (m, 3 H), 7.31 - 7.44 (m, 2 H), 7.82 - 8.09 (m, 1 H) Examples 6 . Amorphous compounds 2 CrystalSince the hemisulfate salt was found to remain solid after 14 days of stability study as shown in Table 6, preliminary tests were conducted to investigate crystallization conditions using 11 different solvents. The amorphous compound was 2Suspended in 5 volumes of solvent (samples 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and 2-11). To those samples that did not flow freely (2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, and 2-10), an additional 5 volumes of solvent were added. Then, each sample was aged at 25-50° C. (1° C./min between temperatures, and 4 hours at each temperature) for 6 days, except for sample 2-1, which was observed as a clear solution after 1 day and evaporated under ambient conditions. The results are shown in Table 9. Crystallization patterns from isobutanol (sample 2-1), acetone (sample 2-2), EtOAc (sample 2-6) and iPrOAc (sample 2-7) was crystallized. Two poorly crystallized samples were also identified from MEK (sample 2-4) and MIBK (sample 2-5) crystals. The XRPD pattern is shown in Figure 7A. surface 9 . Compound 2 Crystallization conditions Sample ID Solvent Observation after 5 volumes Observation after 10 volumes Observation after 1 day aging XRPD 2-1 IPA Solids - Not Free Flowing Free flowing suspension The solution evaporated at RT to produce a gelatinous substance Gel 2-2 Isobutanol Solids - Not Free Flowing Free flowing suspension Suspension Crystallization-Pattern 2 2-3 acetone Solids - Not Free Flowing Free flowing suspension Suspension Crystallization-Pattern 3 2-4 MEK Solids - Not Free Flowing Free flowing suspension Suspension Poor crystallization-Pattern 4 2-5 MIBK Solids - Not Free Flowing Free flowing suspension Suspension Poor crystallization-Pattern 4 2-6 EtOAc Solids - Not Free Flowing Free flowing suspension Suspension Crystallization-Pattern 1 2-7 i Solids - Not Free Flowing Free flowing suspension Suspension Crystallization-Pattern 1 2-8 THF Solids - Not Free Flowing Free flowing suspension Suspension Poor crystallization 2-9 TBME Free flowing suspension - Suspension Amorphous 2-10 Toluene Solids - Not Free Flowing Free flowing suspension Suspension Amorphous 2-11 Heptane Free flowing suspension - Suspension Amorphous Seven samples (samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7 and 2-8) were analyzed by DSC, TGA,¹H-NMR and IC (Table 10, Figure 8A, Figure 8B, Figure 9A, Figure 9B, Figure 10A, Figure 10B, Figure 11A, and Figure 11B), and XRPD analysis after 6 days of storage at 25°C/60% relative humidity (RH) (all samples remained crystalline/poor crystallization after stability). All samples retained approximately half the equivalent of sulfate, but contained relatively large amounts of residual solvent. An overlay of the X-ray diffraction patterns of amorphous samples 2-9, 2-10, and 2-11 is shown in Figure 7B. surface 10 . Crystalline compounds 2 Characteristics of the sample Sample ID Solvent DSC TGA ¹ HNMR IC ( calibration for TGA ) 2-2 Isobutanol Endothermic, 113.8℃ 8.3% Ambient temperature -140℃ 1.1 eq isobutanol 0.45 eq 2-3 acetone Endothermic, 30-95℃ Endothermic, 100-145℃ 7.6% Ambient temperature -140℃ 0.5 eq acetone 0.46 eq 2-4 MEK Wide and complex endothermic, 30-115℃ Endothermic, 115-145℃ 8.5% Ambient temperature -140℃ 0.8 eq MEK 0.45 eq 2-5 MIBK Wide range of heat absorption, 30-105℃ heat absorption, 114.7℃ 5.2% Ambient temperature -110℃ 0.2 eq MIBK 0.46 eq 2-6 EtOAc Sharp endothermic, 113.6℃ 2.0% Ambient temperature -100℃ 0.9 eq EtOAc 0.46 eq 2-7 i Endothermic, 30-90℃ 1.6% Ambient temperature -90℃ 0.8 eq iPrOAc 0.45 eq 2-8 THF Endothermic, 30-100℃ Sharp endothermic, 115.6℃ 4.2% Ambient temperature -130℃ 0.7 eq THF 0.45 eq All samples were subjected to ¹HNMR spectrum, and listed below. Sample 2 - 2 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.83 (d, J=6.69 Hz, 7 H), 0.99 - 1.26 (m, 14 H), 1.61 (dt, J=13.26, 6.63 Hz, 1 H), 3.73 - 3.87 (m, 2 H), 4.03 - 4.18 (m, 1 H), 4.18 - 4.51 (m, 4 H), 4.66 - 4.92 (m, 1 H), 4.70 - 4.90 (m, 1H), 4.72 - 4.88 (m, 1 H), 5.81 (br s, 1 H), 5.93 - 6.11 (m, 2 H), 7.10 - 7.26 (m, 3 H), 7.14 - 7.26 (m, 1 H), 7.30 - 7.41 (m, 2 H), 7.94 (br s, 1H) Sample 2 - 3 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 1.00 - 1.26 (m, 13 H), 2.09 (s, 3 H), 3.74 - 3.87 (m, 2 H), 4.10 (br d, J=7.70 Hz, 1 H), 4.22 - 4.50 (m, 3 H), 4.81 (quin, J=6.28 Hz, 1 H), 5.71 - 5.90 (m, 1 H), 5.96 - 6.15 (m, 2 H), 7.12 - 7.26 (m, 3 H), 7.31 - 7.41 (m, 2 H), 7.79 - 8.07 (m, 1 H) Sample 2 - 4 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.91 (t, J=7.33 Hz, 3 H), 1.01 - 1.28 (m, 13 H), 2.08 (s, 2 H), 3.72 - 3.89 (m, 2 H), 4.10 (br d, J=8.08 Hz, 1 H), 4.23 - 4.47 (m, 3 H), 4.81 (quin, J=6.25 Hz, 1 H), 5.69 - 5.89 (m, 1 H), 5.94 - 6.13 (m, 2 H), 7.14 - 7.25 (m, 3 H), 7.32 - 7.41 (m, 2 H), 7.79 - 8.11 (m, 1 H) Sample 2 - 5 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.86 (d, J=6.69 Hz, 1 H), 0.98 - 1.33 (m, 13 H), 2.02 - 2.09 (m, 1 H), 4.03 - 4.17 (m, 1 H), 4.22 - 4.50 (m, 3 H), 4.81 ( quin, J=6.25 Hz, 1 H), 5.81 (br s, 1 H), 5.93 - 6.15 (m, 2 H), 7.11 - 7.27 (m, 3 H), 7.31 - 7.41 (m, 2 H), 7.77 - 8.21 (m, 1H) Sample 2 - 6 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.98 - 1.28 (m, 15 H), 2.00 (s, 3 H), 3.99 - 4.14 (m, 3 H), 4.21 - 4.49 (m, 3 H), 4.81 (quin, J=6.22 Hz, 1 H), 5.82 (br s, 1 H), 5.93 - 6.14 (m, 2 H), 7.11 - 7.26 (m, 3 H), 7.29 - 7.42 (m, 2 H), 7.79 - 8.17 (m, 1 H) Sample 2 - 7 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.92 - 1.28 (m, 17 H), 1.97 (s, 2 H), 4.04 - 4.16 (m, 1 H), 4.20 - 4.51 (m, 3 H), 4.71 - 4.93 (m, 2 H) , 5.82 (br s, 1 H), 5.95 - 6.14 (m, 2 H), 7.11 - 7.28 (m, 3 H), 7.31 - 7.43 (m, 2 H), 7.75 - 8.21 (m, 1 H) Sample 2 - 8 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.81 - 1.11 (m, 13 H), 1.19 (s, 1 H), 1.53 - 1.66 (m, 1 H), 3.87 - 4.01 (m, 1 H), 4.06 - 4.32 (m, 3 H) , 4.64 (quin, J=6.25 Hz, 1 H), 5.55 - 5.75 (m, 1 H), 5.77 - 5.97 (m, 2 H), 6.94 - 7.10 (m, 3 H), 7.13 - 7.26 (m, 2 H), 7.66 - 7.96 (m, 1H) Examples 7 . Failure to crystallize amorphous malonate( Compound 4) As shown in Example 3, when the compound is tested1The crystalline oxalate can be identified when the appropriate salt is added, but the oxalate compound 4may not be used in clinical trials due to its potential to cause kidney stones. Therefore, attempts were made to crystallize the chemically related malonate salt (compound 5). The compound 1(12 × 50 mg, samples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11 and 3-12) were dissolved in tert-butanol (20 vol), and the solution was then treated with 1 equivalent of malonic acid stock solution (1 M in THF). The sample was then frozen, and in this case the solvent was removed by freeze drying. To samples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10 and 3-11, the relevant solvent (5 vol) was added at room temperature. Any resulting solution was evaporated under ambient conditions, and the gel or solid was aged at 25-50°C (1°C/min between temperatures, and 4 hours at each temperature) for 5 days. The solid was analyzed by XRPD (Figure 12B), but all samples were found to be either gel-like or amorphous (Figure 12B). The results are shown in Table 11. By ¹A solid (amorphous) sample (3-12) was analyzed by H-NMR and HPLC and was found to contain about 1 equivalent of malonic acid (peak overlap) and 0.6 eq.t -BuOH. The compound was 99.2% pure (Figure 13A). Figure 12A is the XRDP of sample 3-12, and Figure 13A is the HPLC chromatogram of sample 3-12. Sample 3 - 12 : ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 0.81 - 1.11 (m, 13 H), 1.19 (s, 1 H), 1.53 - 1.66 (m, 1 H), 3.87 - 4.01 (m, 1 H), 4.06 - 4.32 (m, 3 H) , 4.64 (quin, J=6.25 Hz, 1 H), 5.55 - 5.75 (m, 1 H), 5.77 - 5.97 (m, 2 H), 6.94 - 7.10 (m, 3 H), 7.13 - 7.26 (m, 2 H), 7.66 - 7.96 (m, 1H) surface 11 . Amorphous malonate compound 4 Crystallization conditions Sample ID Solvent Observation after 5 volumes Observation after 5 days of aging / evaporation XRPD 3-1 IPA Clear solution* Transparent gel - 3-2 Isobutanol Clear solution* Transparent gel - 3-3 acetone Clear solution* Transparent gel - 3-4 MEK Clear solution* Transparent gel - 3-5 MIBK Solutions and some gels Transparent gel - 3-6 EtOAc Clear solution* Transparent gel and crystalline form Amorphous 3-7 i Gel Transparent gel - 3-8 THF Clear solution* Transparent gel - 3-9 TBME Viscous suspension Transparent gel - 3-10 Toluene White gel/solid White gel Amorphous 3-11 Heptane White solid (static) White gel Amorphous 3-12 - (white solid-no solvent) (viscous white solid-ambient conditions) Amorphous *Evaporates at room temperature Examples 8 . Liquid-assisted grinding ( LAG ) Failed Form appropriate saltLiquid-assisted grinding (LAG) studies were performed using the 14 acidic counterions in Table 12 to determine appropriate salts other than hemisulfate. surface 12 . LAG Anti-ion stock solution used in crystallization Anti-ion Solvent (1 M) Pyrite DMSO Malonate THF D-Glucuronide water DL-Mandelate THF D-Gluconate THF Glycolate THF L-Lactate THF Oleate THF L-Ascorbic acid water Fatty acid root THF (hot) Hexanoate THF Stearate THF Palmitic Acid THF Methanesulfonate THF Compound1(30 mg) was placed in an HPLC vial with two 3 mm ball bearings. The material was moistened with solvent (15 µl ethanol, samples 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13 and 4-14) and 1 equivalent of acid counter ion was added. The samples were then milled at 650 rpm for 2 hours using a Fritsch milling system with an automatic adapter. Most of the milled samples were found to be clear gels and were not further analyzed (Table 13). Those samples observed to contain solids were analyzed by XRPD, and in all cases the patterns obtained were found to match those of the crystalline acid counterion without the additional peaks (Figure 13B). surface 13 . By compound 1 Of LAG Observations and XRPD Sample ID acid Observation after grinding XRPD 4-1 Acid Yellow gel/solid Pyrrolidone and amorphous halo 4-2 Malonic acid Transparent gel - 4-3 D-Glucuronic acid White gel/solid D-Glucuronic acid and amorphous halo 4-4 DL-Mandelic acid Transparent gel - 4-5 D-Gluconic Acid Transparent gel - 4-6 Glycolic acid Transparent gel 4-7 L-Lactic Acid Transparent gel - 4-8 Oleic acid Transparent gel - 4-9 L-Ascorbic acid White gel/solid L-ascorbic acid and amorphous halo 4-10 Adipic acid Transparent gel - 4-11 Hexanoic acid Transparent gel - 4-12 Stearic acid White gel/solid Stearic acid and amorphous halo 4-13 Palmitic acid White gel/solid Palmitic acid and amorphous halo 4-4 Methanesulfonic acid Transparent gel - Examples 9 . Use of methyl ethyl ketone ( MEK ) Failure to obtain proper salt formationNext, methyl ethyl ketone (MEK) was used as a solvent to investigate suitable salts other than the hemisulfate salt. Using the 14 acid counterions in Table 12, the compounds were 1(50 mg) was dissolved in MEK (20 vol) for investigation. The solution was treated with 1 equivalent of the selected counterion (Table 12). The sample was then cooled to 5°C at 0.1°C/min and stirred at this temperature overnight. All samples were evaporated at ambient conditions and any solids observed were analyzed by XRPD. This evaporation produced mainly gels, except for the samples with stearic acid (samples 4-12) and palmitic acid (samples 5-13), which provided glassy solvents. These solids were all amorphous by XRPD, but no crystalline form of the salt was obtained. The results are shown in Table 14. (Figure 13A). surface 14 . will Compound 1 Dissolve in MEK ( 20 Volume ) The result Sample ID acid Solvent for acids at 1 M Observations after acid addition Observation after cooling down Observation after evaporation 5-1 Acid DMSO Yellow solution Yellow solution Yellow gel 5-2 Malonic acid THF Solution Solution Transparent gel 5-3 D-Glucuronic acid water Solution Solution Transparent gel 5-4 DL-Mandelic acid THF Solution Solution Transparent gel 5-5 D-Gluconic Acid THF White precipitation Turbid solution Transparent gel 5-6 Glycolic acid THF Solution Solution Transparent gel 5-7 L-Lactic Acid THF Solution Solution Transparent gel 5-8 Oleic acid THF Solution Solution Transparent gel 5-9 L-Ascorbic acid water Solution Solution Yellow gel 5-10 Adipic acid THF (hot) Solution Solution Transparent gel 5-11 Hexanoic acid THF Solution Solution Transparent gel 5-12 Stearic acid THF Solution Turbid solution Transparent glassy solid* 5-13 Palmitic acid THF Solution Solution Transparent glassy solid* 5-14 Methanesulfonic acid THF Solution Solution Transparent gel Stock solutions were prepared before acid addition *Samples were analyzed by XRPD and amorphous pattern plus peaks were obtained from acid counterions Since all samples were amorphous, all samples were redissolved in MEK (5 vol) and cyclohexane (20 vol countersolvent) was added at room temperature followed by stirring at 25°C for 1 hour. The samples were then aged between 50°C and 5°C (1°C/min between temperatures, 4 hours at each temperature) for 2 days before the cycle was changed from 50°C to 25°C for another 4 days. The samples were observed by eye after aging. The results are shown in Table 15. After aging, all samples were found to be colloidal except 5-1 (with penic acid). Sample 5-1 (yellow solid) was analyzed by XRPD, and the pattern was found to match the known form of penic acid ( FIG. 14B ), and thus the crystalline form of the salt was not obtained. surface 15 . will Compound 1 Redissolve in MEK ( 5 Volume ) and the results in the antisolvent Sample ID Observe now Observe after 10 minutes 60 minutes of observation Observation after Chenghua 5-1 Sedimentation Gel Gel Yellow suspension** 5-2 Sedimentation Gel Gel Gel 5-3 Sediment/gel Gel Gel Gel 5-4 Sedimentation Gel Gel Gel 5-5 Sediment/gel Gel Gel Gel 5-6 Sedimentation Gel Gel Gel 5-7 Sedimentation Gel Gel Gel 5-8 Sedimentation Light suspension Gel Gel 5-9 Sedimentation Gel Gel Gel 5-10 Sedimentation Gel Gel Gel 5-11 Sedimentation Light suspension Gel Gel 5-12 Sedimentation Light suspension Gel Gel 5-13 Sedimentation Light suspension Gel Gel 5-14 Sedimentation Gel Gel Gel **Sample analyzed by XRPD, where the pattern matches known forms of penic acid (no other peaks) Examples 10 . No adequate salt formation was obtained using ethyl acetateNext, ethyl acetate was used to study suitable salts other than the hemisulfate salt. Using the 14 acid counterions in Table 12, the compounds were heated at 50 °C.1(50 mg) was dissolved in ethyl acetate (20 vol) for this study. The solution was treated with 1 equivalent of the selected counterion (Table 12). The sample was then cooled to 5°C at 0.1°C/min and stirred at this temperature for 4 days. The solution was evaporated at ambient conditions and any solids were analyzed by XRPD. The results of crystallization using ethyl acetate are in Table 16. Compared to Example 8 where MEK was the solvent, most of the sample was observed to be a suspension after cooling of the acid: compound mixture (those in solution were evaporated at ambient conditions). However, the XRPD diffraction patterns were generally found to match the crystalline compound 1. Samples 6-2, 6-4 and 6-5 have some slight differences (Fig. 14A and Fig. 15A). No crystalline form of the salt was obtained. surface 16. will Compound 1 Dissolve in EtOAc ( 20 Volume ) The result Sample ID acid Solvent for acids at 1 M Observations after acid addition Observation after cooling down XRPD Observation after evaporation 6-1 Acid DMSO Yellow solution Yellow solution* - Gel 6-2 Malonic acid THF Solution White suspension Slightly different from free alkali - 6-3 D-Glucuronic acid water Solution Solution* - Gel 6-4 DL-Mandelic acid THF Solution White suspension Slightly different from free alkali - 6-5 D-Gluconic Acid THF White precipitation Possibly white gelatinous substance Slightly different from free alkali - 6-6 Glycolic acid THF Solution White suspension Free base - 6-7 L-Lactic Acid THF Solution White suspension Free base - 6-8 Oleic acid THF Solution White suspension Free base - 6-9 L-Ascorbic acid water Solution Solution* - White solid/yellow gel on the side - amorphous 6-10 Adipic acid THF (hot) Solution White suspension Free base - 6-11 Hexanoic acid THF Solution White suspension Free base - 6-12 Stearic acid THF Solution White suspension Free base - 6-13 Palmitic acid THF Solution White suspension Free base - 6-14 Methanesulfonic acid THF White precipitation Solution/transparent gel* - Gel Examples 11 . By HPLC Of Chemical purity determinationPurity analysis in Examples 2 and 4 was performed on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software vB.04.03 using the method shown in Table 17. surface 17 . Chemical purity test HPLC method Parameters value Method Type Reverse phase with gradient elution Sample preparation 0.5 mg/ml in acetonitrile:water (1:1) String Supelco Ascentis Express C18, 100 × 4.6 mm, 2.7 μm Column temperature (℃) 25 Injection (μl) 5 Wavelength, bandwidth (nm) 255,90 Flow rate (ml/min) 2 Phase A 0.1% TFA/water Phase B 0.085% TFA/acetonitrile Schedule Time(min) Phase A% Phase B% 0 95 5 6 5 95 6.2 95 5 8 95 5 Examples 12 . X Radiation powder diffraction ( XRPD ) TechnologyXRPD patterns for Examples 2, 3, 4, 5, 6, 7, 8, and 9 were collected on a PANalytical Empyrean diffractometer using Cu Kα radiation (45 kV, 40 mA) in a transmission geometry. A 0.5° slit, a 4 mm mask, and a 0.4 rad Soller slit with a focusing lens were used for the incident beam. PIXcel placed on the diffracted beam ^3DThe detector is equipped with a receiving gap and a 0.04 arc Soller gap. The instrument performance is checked on a weekly basis using silicon powder. The software used for data acquisition is X'Pert Data Collector v.5.3, and Diffrac PlusData were analyzed and presented using EVA v.15.0.0.0 or Highscore Plus v.4.5. Samples were prepared and analyzed in transmission mode in metal or Millipore 96-well plates. X-ray transparent film was used between metal sheets on metal well plates, and powders (approximately 1 to 2 mg) were used as received. Millipore plates were used to separate solids from suspensions by adding a small amount of suspension directly to the plate prior to light vacuum filtration and analyzed. Scanning mode for metal plates used an angular scanning axis, while 2θ scanning was used for Millipore plates. Performance checks were performed using silicon powder (metal well plates). The data acquisition details are angular range of 2.5 to 32.0°2θ, step size of 0.0130°2θ and total acquisition time of 2.07 minutes. The samples were also collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a θ-2θ goniometer and V4 divergence and receiving slits, a Ge monochromator and a Lynxeye detector. Instrument performance was verified using a certified corundum standard (NIST 1976). The software used for data acquisition was Diffrac PlusXRD Commander v2.6.1, and using Diffrac PlusData analyzed by EVA v15.0.0.0 and presented. The samples were run at ambient conditions, using powder as-is for flat plate samples. The samples were packaged flat in a cavity cut into a zero-polish background (510) silicon wafer. The sample was rotated in its own plane during analysis. Details of data acquisition were angular range from 2 to 42° 2θ, step size of 0.05° 2θ, and acquisition time of 0.5 sec/step. Examples 13 . Amorphous compounds 2 Synthesis MeOH (151 mL) was added to a 250 mL flask and the solution was cooled to 0-5°C. Concentrated H ₂SO ₄Solution. Add the compound to a separate flask.1(151 g) and acetone (910 mL), and H was added dropwise at 25-30°C over 2.5 hours.₂SO ₄/MeOH solution. A large amount of solid was precipitated. After stirring the solution at 25-30°C for 12-15 hours, the mixture was filtered, washed with MeOH/acetone (25 mL/150 mL), and vacuum dried at 55-60°C to obtain compound 2(121 g, 74%). Compound 2 Analysis Method :The compounds were obtained using an Agilent 1100 HPLC system with a Waters XTerra Phenyl 5 μm 4.6 × 250 mm column under the following conditions 2Purity: 1 mL/min flow rate, reading at 254 nm, 30°C column temperature, 10 μL injection volume and 30 min run time. Samples were dissolved in ACN:water (90:10, v/v). The gradient method used for separation is shown below. Compound 2R _t(min) is approximately 12.0 minutes. Time(min) 0.1% H ₃ PO ₄ /water (A)% Acetonitrile (B)% 0 90 10 20 20 80 20.1 90 10 30 90 10 ¹HNMR: (400 MHz, DMSO- d ₆ ): δ 8.41 (br, 1H), 7.97 (s, 1H), 7.36 (t, J= 8.0 Hz, 2H), 7.22 (d, J= 8.0 Hz, 2H ), 7.17 (t, J= 8.0 Hz, 1H), 6.73 (s, 2H), 6.07 (d, J= 8.0 Hz, 1H), 6.00 (dd, J= 12.0, 8.0 Hz, 1H), 5.81(br, 1H), 4.84-4.73 (m, 1H), 4.44-4.28 (m, 3H), 4.10 (t, J= 8.0 Hz, 2H), 3.85-3.74 (m, 1H), 2.95 (s, 3H), 1.21 (s, J= 4.0 Hz, 3H), 1.15-1.10 (m, 9H). Examples 14 . Compound 2 FeaturesCompounds 2Further by the eyes,¹HNMR, ¹³CNMR, ¹⁹FNMR, MS, HPLC and XRPD (Figure 15B) characterization. Residual solvent was measured by GC. Water content was measured by Karl Fischer titration and was only 0.70%. The data are summarized in Table 18. surface 18 . Compound 2 Overview of additional feature data Test result Form White solid NMR ¹ HNMR peaks are listed in Example 4 MS MS (ESI+ve) [M+H] ⁺ = 582.3, consistent with the structure HPLC By AUC at 254 nm, 99.8% (average of two preparations) By residual solvent in GC Methanol, 57 ppm Acetone, 752 ppm Methylene chloride, 50 ppm Ethyl acetate, 176 ppm Water content 0.70% Examples 15 . Compound 1 and compounds 2 SolubilityAll tested compounds 1and compounds 2Solubility in biorelevant test media, including simulated gastric fluid (SGF), simulated fasted gastric fluid (FaSSIF), and satiated gastric fluid (FeSSIF). Compounds 1The results are shown in Table 19, and the compound 2The results are shown in Table 20. The samples were stirred at room temperature (20-25°C). Compound 2Compounds 1More than 40 times more soluble in water at 2 hours and more than 25 times more soluble at 24 hours. In SGF conditions, compared with the compound at the same time point 1Compared with the solubility of 15.6 mg/mL, the compound 2It has a solubility of 84.2 mg/mL at 24 hours. Under SGF conditions, compound 2Also at 2 hours, the compound1More soluble, and soluble enough to allow testing even after 48 hours, while compounds 1No testing was performed at 48 hours. surface 19 . Compound 1 Solubility test results Test medium Solubility ( in mg /mL ) Form Descriptive terms 2 hours 24 hours water 1.5 2.5 Clear solution* Slightly soluble SGF 13.8 15.6 Clear solution with a gel at the bottom Slightly soluble FaSSIF 1.7 1.7 Turbid Slightly soluble FeSSIF 2.8 2.9 Turbid Slightly soluble *The sample appeared clear and only achieved a solubility of 1.5 mg/mL. Upon further investigation, it was noted that a gelatinous film formed on the stir bar. Compound 1 active ingredient formed gelatinous spheres in the diluent (90% water/10% acetonitrile) during the standard preparation, which required longer sonication time for complete dissolution. surface 20 . Compound 2 Solubility test results Test medium Solubility ( mg /mL salt ) Form Descriptive terms 2 hours 24 hours 48 hours water 65.3 68.0 N/A Turbid Soluble SGF 89.0 84.2 81.3 Turbid Soluble FaSSIF 1.9 2.0 N/A Turbid Slightly soluble FeSSIF 3.3 3.4 N/A Turbid Slightly soluble Examples 16 . Compound 2 Chemical stabilityBy monitoring the organic purity, water content,¹HNMR, DSC and Ramen IR to test compounds 2Chemical stability of the container closure system used in the study was a combination pharmaceutical valve bag with a pharmaceutical laminate film on the bag and a dry silicone between the two layers. Weighing the compound 2(1 g) into each container. The bags were then stored at 25°C/60% RH (relative humidity) and 40°C/75% RH (relative humidity). Organic purity, water content, ¹HNMR, DSC and Raman spectra. The compounds were obtained using a Shimadzu LC-20AD system with a Waters XTerra Phenyl 5 μm 4.6 × 250 mm column under the following conditions 2Purity: 1 mL/min flow rate, read at 254 nm, 35°C column temperature and 10 μL injection volume. Samples were dissolved in acetonitrile-water (90:10) (v/v). The gradient method is shown below. Time (min) A% (ACN) B% ( water ) 0 90 10 20 20 80 20.1 90 10 30 90 10 Determination of compounds using Karl Fischer titration with water titration equipment 2(250 mg) of water content. The results are shown in Table 21 and Table 22. When the compound 2When stored at 25°C and 40°C for 6 months, the degradation rate was minimal. At 3 months, the compound 299.75% pure at 25°C and 99.58% pure at 40°C. At 6 months, the compound 2It is still 99.74% pure at 25°C and 99.30% pure at 40°C. At 25°C, the percentage of degradation products increases from 0.03% on day 0 to 0.08% after 6 months. At 40°C, the percentage of degradation products increases from 0.03% to 0.39%. Over the course of 6 months, the percentage of water increases by approximately 0.6% at 25°C and approximately 0.7% at 40°C. Compound 2At 1, 2, 3 and 6 months,¹Characteristics and compounds of HNMR, Raman spectroscopy and DSC 2The characteristics at day 0 were identical under both temperature conditions (Table 22), highlighting the compound 2Long-term stability. surface twenty one . Compound 2 exist 6 Within a month 25 ℃ and 40 ℃ The degradation rate Test time Water percentage Purity percentage Percentage of degradation products Maximum impurity percentage 25 ℃ Day 0 1.2 99.82 0.03 0.12 1st month 1.9 99.77 0.04 0.12 Second month 1.8 99.75 0.06 0.12 3rd Month 1.8 99.75 0.06 0.12 6th month 1.8 99.74 0.08 0.13 40 ℃ Day 0 1.2 99.82 0.03 0.12 1st month 2.0 99.71 0.09 0.12 Second month 1.9 99.63 0.15 0.12 3rd Month 1.9 99.58 0.20 0.12 6th month 1.9 99.30 0.39 0.14 surface twenty two . Compound 2 Characteristics during degradation studies Test time ¹ HNMR Raman DSC 25 ℃ Day 0 Test Start Test Start Test Start 1st month Same as Day 0 Same as Day 0 Same as Day 0 Second month Same as Day 0 Same as Day 0 Same as Day 0 3rd Month Same as Day 0 Same as Day 0 Same as Day 0 6th month Same as Day 0 Same as Day 0 Same as Day 0 40 ℃ Day 0 Test Start Test Start Test Start 1st month Same as Day 0 Same as Day 0 Same as Day 0 Second month Same as Day 0 Same as Day 0 Same as Day 0 3rd Month Same as Day 0 Same as Day 0 Same as Day 0 6th month Same as Day 0 Same as Day 0 Same as Day 0 Measuring compounds 2Additional chemical stability studies were conducted to determine the impurities and water content. Three conditions were tested: accelerated stability over a 6-month period (40 ± 2°C / 75 ± 5% RH); environmental stability over a 9-month period (25 ± 2°C / 60 ± 5% RH); and stability under freezer conditions (5 ± 3°C) over a 9-month period. The results of accelerated stability, environmental stability, and freezer conditions are shown in Tables 23, 24, and 25, respectively. Based on the results of these studies, compound 2is polarically stable. In the accelerated stability study (Table 23), the measured compounds 2At each time point (1st month, 3rd month and 6th month), the compound 2The form of the product was always a white solid and the IR was consistent with the reference standard. After six months, the total impurity of related substance 1 was only 0.08% and related substance 2 and isomers were not detected. surface twenty three . Compound 2 Accelerated stability ( 40 ± 2 ℃ / 75 ± 5 % RH ) Items Specifications Test time Month 0 1st month 3rd Month 6th month Form White or off-white solid White solid White solid White solid White solid IR Corresponding to the reference standard Corresponding to the reference standard / Corresponding to the reference standard Corresponding to the reference standard water ≤2.0% 0.45% 0.21% 0.36% 0.41% Related substances 1 Impurity A ≤0.15% ND ND ND ND Impurity B ≤0.15% ND ND ND ND Impurity F ≤0.15% ND ND ND 0.01% Impurity H ≤0.15% ND ND ND ND Any other single impurity ≤0.10% 0.01% 0.02% 0.01% 0.05% Total impurities ≤0.2% 0.01% 0.02% 0.02% 0.08% Related substances 2 Impurity G ≤0.15% ND ND ND ND Isomers Impurity C ≤0.15% ND / ND ND Impurity D ≤0.15% ND / ND ND Impurity E ≤0.15% ND / ND ND analyze 98.0%~102.0% 98.8% 101.5% 99.6% 99.5% Microbiological testing TAMC ≤1000cfu/g ＜1cfu/g / / / Mold and Yeast ≤100cfu/g ＜1cfu/g / / / Escherichia coli Not detected ND / / / N.D.: Not Detected In a nine-month environmental stability study measuring speciation, IR, water, and impurity content, the compound 2The morphology of the compound is always a white solid and the IR always corresponds to the reference sample. The results (Table 24) emphasize that the compound 2How chemically stable. After 9 months, the percentage of water in the sample was only 0.20% and the total relevant substance 1 impurity was only 0.02%. Similar to the accelerated stability study, relevant substances 2 and compounds were not detected. 2Any isomers of. surface twenty four . Compound 2 Environmental stability ( 25 ± 2 ℃ / 60 ± 5 % RH ) Items Specifications Test time Month 0 1st month 3rd Month 6th month 9th month Form White or off-white solid White solid White solid White solid White solid Off-white solid IR Corresponding to the reference standard Corresponding to the reference standard / Corresponding to the reference standard Corresponding to the reference standard Corresponding to the reference standard water ≤2.0% 0.45% 0.19% 0.29% 0.46% 0.20% Related substances 1 Impurity A ≤0.15% ND ND ND ND ND Impurity B ≤0.15% ND ND 0.03% ND ND Impurity F ≤0.15% ND ND 0.02% 0.01% ND Impurity H ≤0.15% ND ND ND ND ND Any other single impurity ≤0.10% 0.01% 0.01% 0.03% 0.02% 0.02% Total impurities ≤0.2% 0.01% 0.02% 0.11% 0.05% 0.02% Related substances 2 Impurity G ≤0.15% ND ND ND ND ND Isomers Impurity C ≤0.15% ND / ND ND ND Impurity D ≤0.15% ND / ND ND ND Impurity E ≤0.15% ND / ND ND ND analyze 98.0%~102.0% 98.8% 101.1% 99.6% 99.7% 100.9% Microbiological testing TAMC ≤1000cfu/g ＜1cfu/g / / / / Mold and Yeast ≤100cfu/g ＜1cfu/g / / / / Escherichia coli Not detected ND / / / / N.D.: Not Detected The results of stability measurements under freezer conditions are shown in Table 25. The only impurities detected even after 9 months were those from related substance 1 and water. The water content after 9 months was 0.32% and the total impurities of related substance 1 were only 0.01% of the sample. Compound 2 is extremely chemically stable under freezer conditions. surface 25 . In freezer conditions ( 5 ± 3 ℃ ) The following compounds 2 Stability Items Specifications Test time Month 0 1st month 3rd Month 6th month 9th month Form White or off-white solid White solid White solid White solid White solid Off-white solid IR Corresponding to the reference standard Corresponding to the reference standard / Corresponding to the reference standard Corresponding to the reference standard Corresponding to the reference standard water ≤2.0% 0.45% 0.19% 0.32% 0.42% 0.32% Related substances 1 Impurity A ≤0.15% ND ND ND ND ND Impurity B ≤0.15% ND ND 0.01% ND ND Impurity F ≤0.15% ND ND ND ND ND Impurity H ≤0.15% ND ND ND ND ND Any other single impurity ≤0.10% 0.01% 0.01% 0.01% 0.01% 0.01% Total impurities ≤0.2% 0.01% 0.01% 0.03% 0.03% 0.01% Related substances 2 Impurity G ≤0.15% ND ND ND ND ND Isomers Impurity C ≤0.15% ND / ND ND ND Impurity D ≤0.15% ND / ND ND ND Impurity E ≤0.15% ND / ND ND ND analyze 98.0%~102.0% 98.8% 101.1% 100.2% 98.6% 101.4% Microbiological testing TAMC ≤1000cfu/g ＜1cfu/g / / / / Mold and Yeast ≤100cfu/g ＜1cfu/g / / / / Escherichia coli Not detected ND / / / / N.D.: Not detected Examples 17 . In a single oral dose of the compound 2 Plasma levels of metabolitesA single oral dose of the compound was administered to rats, dogs, and monkeys.2, and the plasma levels of certain metabolites shown in Scheme 1 were measured. Compounds 2Transformed into compounds 1and metabolites 1 - 7The results for metabolites 1-8 and 1-2 are shown in Table 26 and Table 27. In rats, lower levels of compound 1exposure, but higher levels of metabolites were observed 1 - 7, which is an active triphosphate (metabolite 1 - 6) nucleoside metabolite. In monkeys, the compound was measured in approximately dose-proportional amounts 1In dogs, overproportionate amounts of the compound were measured1exposure, which indicates first-pass metabolic clearance in the liver. Throughout the study, significantly more vomiting was observed in dogs (5/5 higher dose group) than in monkeys (1/5 higher dose group). surface 26 . In a single oral dose of the compound 2 Post-compound 1 and metabolites 1 - 7 Plasma level Species Dosage* (mg/kg) Compound 1 Metabolites 1-7 C _max (ng/mL) _Tmax (hr) AUC _0-last (hr*ng/mL) C _max (ng/mL) AUC _0-last (hr*ng/mL) ^Rat 500 70.5 0.25 60.9 748 12000 ^Dog 30 1530 0.25-1 1300 783 9270 100 8120 0.5-1 10200 2030 24200 300 21300 204 44300 4260 60800 ^Monkey 30 63.5 0.5-2 176 42.5 1620 100 783 1-2 1100 131 3030 300 501 204 1600 93.6 3660 3 males/doses/species; *Dosage formula: ^a0.5% CMC, water containing 0.5% Tween 80; ^bCapsules containing powder surface 27 . In a single oral dose of the compound 2 Descendants 1 - 8 and 1 - 2 Plasma level Species Dosage* (mg/kg) Metabolites 1-8 Metabolites 1-2 C _max (ng/mL) AUC _0-last (hr*ng/mL) C _max (ng/mL) AUC _0-last (hr*ng/mL) ^Rat 500 5060 35100 9650 20300 ^Dog 30 291 905 196 610 100 1230 4370 886 2830 300 5380 35300 2380 8710 ^Monkey 30 209 5690 300 1730 100 406 12300 1350 8160 300 518 16800 1420 11400 3 males/doses/species; *Dosage formula: ^a0.5% CMC, water containing 0.5% Tween 80; ^bCapsules containing powder Examples 18 . Compound 2 Tissue exposure of active triphosphates after oral dosingCompounds in oral doses 2Compounds were measured 4 hours later 2Active triphosphate (TP) (metabolite 1 - 6) levels in heart and liver tissues. In a single dose of the compound 2Liver and heart samples were obtained 4 hours after the experiment, snap frozen, homogenized, and analyzed by LC-MS/MS for intracellular active TP levels. Tissue levels were measured in rats, dogs, and monkeys, as shown in Figure 16A. High levels of active TP were measured in the liver of all species tested. Relatively low levels of active TP were measured in the heart of dogs, due to saturation of first-pass liver metabolism, and incalculable levels of TP were measured in the hearts of rats and monkeys, indicating liver-specific formation of active TP. Although not shown, the same compounds 1Compared with drug administration, the compound 2Improved TP distribution through medication administration. Examples 19 . Compounds in Dogs 1 Compounds 2 Comparative pharmacologyCompounds for use 1and compounds 2A head-to-head comparison of dogs given the drug. The study measured the compound used in the study.1(25 mg/kg) and compounds 2(30 mg/kg) Compound 4 hours after administration 1and metabolites 1 - 7(from Process 1) plasma levels (Table 28), and metabolites 1 - 7AUC _{( 0 - 4hr )}In the compound 2and compounds 1In comparison, it is twice as large. Compound 1and metabolites 1 - 7The dose-normalized exposures are shown in Table 28. Compounds 1, metabolites 1 - 7and compounds 1+ Metabolites 1 - 7AUC of the sum of_{( 0 - 4hr )}The value of the compound in the administration of2Later higher. surface 28 . Compounds in use 1 and compounds 2 Comparison of plasma levels after drug administration Compounds for administration Dose-normalized mean AUC _{(0 -4hr )} (μM × hr ) for : Compound 1 Metabolites 1-7 Compound 1 + metabolites 1-7 Compound 1 (25 mg/kg) 0.2 1.9 2.1 Compound 2 (30 mg/kg) 1.0 4.1 5.1 ^aAUC _{(0 -4hr )}Values normalized to a dose of 25 mg/kg Liver/heart ratios indicate triphosphate concentrations, and compounds 1In contrast, using compounds 2Administration increased the selective delivery of triphosphate to the liver as shown in Table 29. Administered compounds measured in the heart 1The active guanine metabolite ( 1 - 6)'s AUC _{( 0 - 4hr )}is 174 μM × hr, while the administered compound measured in the heart 2The active guanine metabolite ( 1 - 6)'s AUC _{( 0 - 4hr )}is 28 μM × hr. With compound 1Compared with a liver/heart ratio of 3.1, compound 2The liver/heart ratio is 20. surface 29 . Compounds 1 and compounds 2 Comparison of posterior liver and heart exposure Compounds for administration Dose-normalized mean AUC _{(0 -4hr )} (μM × hr ) for : liver Heart Liver/heart Compound 2 565 28 ^b 20 Compound 1 537 174 3.1 ^aActive TP concentration ( 1 -6; Process 1) Standardized to a dose of 25 mg/kg ^bExtrapolation below the lower limit of the quantitative calibration curve When compared with compounds 1Compared to administration of compounds 2The effect of increasing the selectivity for liver over heart is also shown in Figure 16B. When the compound is administered2The levels of active triphosphate in heart and liver tissues after administration of compound (30 mg/kg) were similar to those after administration of compound 1(25 mg/kg) compared to the tissue levels of active triphosphates. For compound 1and compounds 2In general, the concentration of active TP in the liver is higher than that in the heart, but when combined with compounds 1Compared to the administered compound 2Active TP is more selective for the liver than the heart. Examples 20 . Compounds in rats and monkeys 2 Metabolite plasma curveMale Sprague-Dawley rats and cynomolgus monkeys (3 animals/dose group) were given a single oral dose of the compound 2. Analysis of compounds in plasma aliquots prepared from dichloroisothiazide-treated blood samples by LC-MS/MS 1and metabolites 1 - 7(Compounds shown in Scheme 1 2The concentrations of active triphosphate nucleoside metabolites of α-glucose were measured in rats and the pharmacokinetic parameters were determined using WinNonlin. The results of a single 500 mg/kg dose in rats are shown in Figure 17, and the results of a single 30 mg/kg, 100 mg/kg, or 300 mg/kg dose in monkeys are shown in Figure 18. The results are summarized in Table 30. Metabolites at higher plasma levels 1 - 7(Compounds 2The results of the study showed that the active triphosphate (TP) nucleoside metabolite of TP was formed, indicating that higher levels of TP were formed even in rats where extremely low plasma levels of the parent nucleotide prodrug were observed, which was due to the compound 1Short half-life in rat blood (<2 min). Persistent plasma levels of metabolites 1 - 7Reflecting the longer half-life of TP. In monkeys, the compound 1The plasma exposure (AUC) of levofloxacin is roughly proportional to the dose, while the metabolites 1 - 7Exposure was slightly disproportionate to dose, but AUC values for both parent drug and the nucleoside metabolite of active TP continued to increase up to the highest dose tested (300 mg/kg). Oral administration of compounds in rats and monkeys 2Produces higher and dose-dependent plasma exposure and metabolites 1 - 7(Compounds 2Nucleoside metabolites of intracellularly active triphosphates); metabolites 1 - 7Exposure continued to increase up to the highest dose tested, reflecting substantial formation of active TP in these species. surface 30 . In a single oral dose of the compound 2 Post-compound 1 and 1 - 7 Plasma level Species ^{Rat Monkey} Dosage (mg/kg) 500 30 100 300 Compound 1 C _max (ng/mL) 60.8 63.5 783 501 _Tmax (hr) 0.25 0.5-2 1-2 204 AUC _0-last (hr*ng/mL) 78.2 176 1100 1600 Metabolites 1-7 C _max (ng/mL) 541 42.5 131 93.6 AUC _0-last (hr*ng/mL) 9640 1620 3030 3660 _Tmax (hr) 6-8 12-24 4 4-24 T _1/2 (hr) 15.3 11.5 15.0 18.8 Dosage formula: ^a0.5% CMC, water containing 0.5% Tween 80; ^bCapsules containing powder Examples twenty one . Compound 1 and compounds 2 Effects of active triphosphates on mitochondrial integrityThe compound will be incorporated into human mitochondrial RNA polymerase 1and compounds 2Active triphosphate (TP) (metabolite 1 - 6The relative potency of (Scheme 1)) is compared with the relative potency of the active TP of sofosbuvir and the active TP of INX-189. Compound 1and compounds 2Unlikely to affect mitochondrial integrity due to its poor incorporation of the active triphosphate by human mitochondrial RNA polymerase, with efficacy similar to that of the triphosphate of sofosbuvir; the relative efficacy of the triphosphate incorporated into INX-189 was up to 55-fold higher. Results are shown in Table 31. According to Arnold et al. (Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleotides. PLoS Pathog ., 2012, 8, e1003030) to measure the incorporation of these analogs by human mitochondrial RNA-dependent RNA polymerase (POLRMT). surface 31 . use Human mitochondria RNA Polymerase Kinetic parameters of evaluated nucleotide analogs Nucleotide analogs K _pol (s ^-1 ) K _d,app (μM) K _{pol/K d,app} (μM ^-1 s ^-1 ) Relative efficacy * 2' - Deoxy - 2'-F - 2'-C -methylUTP ( the active TP of sofosbuvir ) 0.00034 + 0.00005 590 + 250 5.8 x ^10-7 + 2.6 x ^10-7 1.0 ^x10-6 2' -C - methyl GTP ( active TP of INX -189 ) 0.051 + 0.002 240 + 26 2.1 x ^10-4 + 0.2 x ^10-4 5.5 ^x10-5 Active TP of Compound 1 and Compound 2 ( metabolites 1-6 ) 0.0017 + 0.0002 204 + 94 8.3 x ^10-6 + 4.0 x ^10-6 2.2 ^x10-6 *Relative efficacy = (K _pol/K _{d ,app})Nucleotide analogs/(K _pol/K _{d ,app})Natural nucleotides Examples twenty two . Compound 1 For NS5B Activity of the replicator of the sequenceA set of replicons containing NS5B sequences from various HCV genotypes derived from 6 laboratory reference strains (GT1a, GT1b, GT2a, GT3a, GT4a, and GT5a) (Figure 19) and from 8 HCV patient plasma samples (GT1a, GT1b, GT2a, GT2b, GT3a-1, GT3a-2, GT4a, and GT4d) (Figure 20) were used to test compounds 1and concentrations of sofosbuvir. For clinical and laboratory strains of HCV, the compound 1More potent than sofosbuvir. Against wild-type clinical isolates, the compound 1Demonstrated potent pan-genotypic antiviral activity in vitro, with EC ₉₅＜ 80 nM, which is 4 to 14 times more potent than sofosbuvir. As shown in Figure 20, against all clinical isolates of HCV genotypes tested, compound 1EC ₉₅The values were 7 to 33 times lower than those of sofosbuvir. For laboratory strains of HCV genotypes 1-5, the compound 1EC ₅₀The values were 6 to 11 times lower than those of sofosbuvir (Figure 19).Examples twenty three . Healthy Volunteers ( part A ) and GT1 - HCV Infected patients ( part B ) Zhongzhi Compound 2 Single bolus dose ( SAD ) ResearchStudying test compounds at single ascending doses (SADs) 2To measure its safety, tolerability and pharmacokinetics in healthy subjects (Part A). Part A is a randomized, double-blind, placebo-controlled SAD study. Healthy subjects in Part A received a single dose of the compound in a fasting state.2or placebo. Subjects were confined to the clinic from day 1 to day 6. Dosing was staggered across groups so that 2 subjects (1 active: 1 placebo) were evaluated 48 hours after dosing before dosing the rest of the group. Groups received compounds in ascending order 2. Dosing of sequential cohorts was performed based on review of available safety data (through day 5) and plasma pharmacokinetic data (through 24 h) from previous cohorts. Dose escalation was performed following satisfactory review of these data. The doses evaluated in cohorts 3a to 4a were adjusted in increments of no greater than 100 mg as pharmacokinetic and safety data emerged from previous cohorts. The total maximum dose evaluated in Part A did not exceed 800 mg. The dosing regimen for Part A is shown in Table 32. surface 32 . part A Administration of compound 2 Study dosing regimen Group group N ( Active : Placebo) Compound 2 ( Compound 1 ) * 1a healthy 6:2 50 (45) mg × 1 day 2a healthy 6:2 100 (90) mg × 1 day 3a healthy 6:2 200 (180) mg × 1 day 4a healthy 6:2 400 (360) mg × 1 day *About compounds 2Clinical doses are expressed as approximate compound 1 base equivalents in parentheses Healthy volunteers in Part A of the study were male and female individuals between 18 and 65 years of age. Active and placebo recipients were pooled within each Part A group to maintain study blinding. Test compounds were also studied as single ascending doses (SADs) 2, to measure its safety, tolerability, pharmacokinetics and antiviral activity in GT1-HCV infected patients (Part B). Subjects in Part B received a single dose of the compound in a fasting state 2. Confine patients to the clinic from day 1 to day 6. Part B begins after review of safety (through day 5) and plasma pharmacokinetic (through 24 h) data from cohort 3a in part A. Review available safety data (through day 5) and pharmacokinetic data (through 24 h) from the first cohort in part B (cohort 1b) prior to enrolling subsequent part B cohorts. Subsequent part B cohorts are dosed only after review of available safety and pharmacokinetic data from individual doses in part A and available safety (through day 5) from the previous part B cohort. After satisfactory review of these data, dose escalation up to 600 mg is performed in HCV-infected patients. The dosing regimen for Part B is shown in Table 33. surface 33 . part B Compounds in 2 Study dosing regimen Group group N ( Active) Compound 2 ( Compound 1 ) * 1b GT1 HCV infection 3 100 (90) mg × 1 day 2b GT1 HCV infection 3 300 (270) mg × 1 day 3b GT1 HCV infection 3 400 (360) mg × 1 day 4b GT1 HCV infection 3 600 (540) mg × 1 day *About compounds 2Clinical doses are expressed as the approximate compound 1 basis equivalents in parentheses. Patients infected with HCV have ≥ 5 log ₁₀IU/mL viral load in untreated, non-cirrhotic GT1-infected individuals. No serious adverse signs were recorded, and no premature discontinuation was required in either Part A or Part B. All adverse effects were mild to moderate in intensity with no dose-related pattern, including laboratory parameters, vital signs, and ECG were unremarkable. Examples twenty four . Compound 2 Single bolus dose ( SAD ) Results of the studySingle dose of compound 2Post-measurement compounds 1and nucleoside metabolites 1 - 7Pharmacokinetics of the compound at a dose of 600 mg 2Metabolites in post-HCV infected patients 1 - 7C _{twenty four}Minimum plasma concentration (C _24h) is 25.8 ng/mL, which is a 300 mg dose of the compound 2Plasma concentration after dose more than doubles. Metabolites 1 - 7(Shown in Scheme 1) can be achieved simply by dephosphorylating intracellular phosphate metabolites 1 - 4, metabolites 1 - 5and metabolites 1 - 6produced, which is the active species. Therefore, metabolites 1 - 7Can be considered as a substitute for the active species. The pharmacokinetic data of all groups are shown in Tables 34 and 35. Except T _maxAll values are reported as mean ± SD, except for values reported as median (range). Pharmacokinetic parameters were similar in healthy and HCV-infected patients. surface 34 . A single dose of the compound was administered to healthy volunteers 2 Subsequent compounds 1 and metabolites 1 - 7 Human Pharmacokinetics Dosage (mg) C _max (ng/mL) _Tmax (h) AUC _tot (ng*h/mL) T _1/2 (h) C _24h (ng/mL) Part A , Healthy Individuals Compound 1 50 46.4 ±17.6 0.5 (0.5-0.5) 36.4 ± 12.3 0.32 ± 0.02 -- 100 156 ± 96.3 0.5 (0.5-1.0) 167 ± 110 0.53 ± 0.24 -- 200 818 ± 443 0.5 (0.5-3.0) 656 ± 255 0.71 ± 0.16 -- 400 1194 ± 401 0.5 (0.5-1.0) 1108 ± 326 0.86 ± 0.15 -- Metabolites 1-7 50 27.9 ± 5.62 3.5 (3.0-4.0) 285 ± 69.4 7.07 ± 4.59 2.28 ± 0.95 100 56.6 ± 14.0 4.0 (3.0-6.0) 663 ± 242 17.7 ± 14.7 4.45 ± 1.87 200 111 ± 38.8 5.0 (3.0-6.0) 1524 ± 497 15.9 ± 7.95 13.7 ± 5.09 400 153 ± 49.4 6.0 (4.0-8.0) 2342 ± 598 15.6 ± 6.37 23.5 ± 6.31 *Based on 24-hr curve. surface 35 . exist GT1 HCV Administration of compounds to infected patients 2 Subsequent compounds 1 and metabolites 1 - 7 Human Pharmacokinetics Dosage (mg) C _max (ng/mL) _Tmax (h) AUC _tot (ng*h/mL) T _1/2 (h) C _24h (ng/mL) Compound 1 100 212 ± 32.0 0.5 (0.5-1.0) 179 ± 54.4 0.54 ± 0.12 -- 300 871 ± 590 0.5 (0.5-1.0) 818 ± 475 0.64 ± 0.20 -- 300 2277 ± 893 0.5 (0.5-1.0) 1856 ± 1025 0.84 ± 0.18 -- 400 2675 ± 2114 1.0 (1.0-2.0) 2408 ± 1013 0.86 ± 0.18 -- 600 3543 ± 1649 1.0 (0.5-1.0) 4132 ± 1127 0.70 ± 0.13 -- Metabolites 1-7 100 50.2 ± 15.4 6.0 (4.0-6.0) 538 ± 103* 8.4 ± 4.3* 3.60 ± 0.40 300 96.9 ± 38.9 6.0 (3.0-6.0) 1131 ± 273* 8.1 ± 2.4* 10.9 ± 3.51 300 123 ± 16.6 4.0 (3.0-6.0) 1420 ± 221 -- 18.0 ± 8.83 400 160 ± 36.7 4.0 (4.0-4.0) 2132 ± 120 11.6 ± 1.21 22.5 ± 3.29 600 198 ± 19.3 4.0 (4.0-6.0) 2176 ± 116 -- 25.8 ± 4.08 *Based on 24-hr curves. Also calculated for compounds in all groups of Part A and Part B of the study 1and metabolites 1 - 7Figure 21 shows the average plasma concentration-time curve of the compound after a single dose.2Post-compound 1The average plasma concentration of, and Figure 22 is the compound in a single dose 2Descendants of the past1 - 7As shown in Figure 21, compound 1Rapidly absorbed and rapidly/amply metabolized in all groups from fraction B. As shown in Figure 22, metabolites 1 - 7It is the main metabolite and presents a stable plasma concentration. Compound 1Plasma exposure is dose-related, while metabolites 1 - 7The exposure was dose proportional. For HCV-infected individuals in Part B, after administration of the compound 2HCV RNA was quantitatively measured before, during, and after. Plasma HCV RNA was measured using a validated commercial assay. Baseline was defined as the mean of Day-1/Day 1 (pre-dose). A single 300 mg dose of compound 2(equivalent to 270 mg compound 1) produced significant antiviral activity in GT1b-HCV infected individuals. The mean maximum HCV RNA reduction 24 hours after administration following a single 300 mg dose was 1.7 log ₁₀IU/mL and compared this to a 2 log10 IU/mL reduction after 1 day of sofosbuvir monotherapy at 400 mg in GT1a HCV-infected individuals. The mean maximum HCV RNA reduction 24 hours after a single 100 mg dose was 0.8 log ₁₀IU/mL. The mean maximum HCV RNA reduction after a single 400 mg dose was 2.2 log ₁₀IU/mL. Individual pharmacokinetic/pharmacodynamic analyses of individual subjects from Part B of the study are shown in Figures 23A to 23F. Plotting metabolites 1 - 7The concentrations were targeted to reduce the HCV RNA concentrations, and as shown in Figures 23A to 23F, the plasma HCV RNA reduction was associated with plasma metabolites 1 - 7Exposure related. Viral response is greater than EC against GT1b ₉₅Metabolites of value 1 - 7Plasma concentrations were maintained. The correlation between plasma concentrations and the level of HCV RNA reduction indicated that higher doses of the compound were effective.2A more in-depth response should be achieved. Examples 25 . For HCV GT 1 - 4 More than one compound of clinical isolates 1 EC ₉₅ Metabolites of value 1 - 7 The lowest level of stable forecastAs shown in Figure 24, the compound is predicted to be administered in humans 2(600 mg QD (550 mg free base equivalent) and 450 mg QD (400 mg free base equivalent)) 1 - 7The stable minimum plasma level (C _{twenty four , ss}) and all compounds in the tested clinical isolates in vitro 1EC ₉₅Comparison is used to determine whether the steady-state plasma concentration is always higher than EC ₉₅, which will produce increased efficacy in vivo against any or all clinical isolates tested. Compounds 1EC ₉₅and compounds 2EC ₉₅Same. For effective compounds 2, metabolites 1 - 7The stable minimum plasma level should exceed EC ₉₅. As shown in Figure 24, the compounds against all tested clinical isolates 2EC ₉₅The range is approximately 18 to 24 nM. As shown in Figure 24, the compound in humans at a dose of 450 mg QD (400 mg free base equivalent) 2Provides a predicted steady-state minimum plasma concentration of approximately 40 ng/mL (C _{twenty four , ss}). Compounds present in humans at a dose of 600 mg QD (550 mg free base equivalent) 2Provides a predicted steady-state minimum plasma concentration of approximately 50 ng/mL (C _{twenty four , ss}). Therefore, substitute metabolites 1 - 7The predicted steady-state plasma concentration is the EC for all clinical isolates tested (even the difficult GT3a)₉₅, which indicates excellent potency. In contrast, the EC of the standard of the nucleotide sofosbuvir ₉₅The EC values of all HCV clinical isolates tested ranged from 50 to 265 nM, with only two isolates, GT2a and GT2b, having EC values higher than 2.₉₅Less than the predicted steady-state concentration at the commercial dose of 400 mg. For other clinical isolates GT1a, GT1b, GT3a, GT4a, and GT4d, the EC of the commercial dose of 400 mg sofosbuvir ₉₅Greater than predicted steady-state concentration. Using 300 mg minimum steady-state plasma concentration (C_{twenty four , ss})Predicted compounds 2450 mg Steady-state minimum plasma concentration (C _{twenty four , ss}). Average steady-state minimum plasma concentration at 300 mg (C _{twenty four , ss}) is 26.4 ng/mL, and the calculated value is therefore 26.4×450/300=39.6 ng/mL. Use the following three methods to predict the 600 mg steady-state minimum plasma concentration (C _{twenty four , ss}): 1) 600 mg on day 1 C _{twenty four}The mean value was 25.8 ng/mL and a 60% increase was assumed to reach steady state. Therefore, the calculated value is 25.8×1.6=41.3 ng/mL; 2) 400 mg Day 1 C _{twenty four}The mean value was 22.5 ng/mL and a 60% increase was assumed to achieve steady state. Considering dose proportional PK, the calculated value was 22.5×1.6×600/400=54 ng/mL; and 3) 300 mg steady state minimum plasma concentration (C _{twenty four , ss}) is 26.4 ng/mL and proportional PK is assumed. Therefore, the calculated value is 26.4×2=52.8 ng/mL. 600 mg Steady-state minimum plasma concentration (C _{twenty four , ss}) is the average of 3 data points ( (41.3+54+52.8)/3=49.3 ng/mL). Compared with C after a single dose _{twenty four}In contrast, in a stable state, C _{twenty four}There is usually about a 60% increase in the presence of. Comparison of the efficacy and pharmacokinetic stability parameters in Figure 24 clearly shows that the compounds used to treat hepatitis C 2of unexpected therapeutic importance. In fact, administration of the compound 2The predicted steady-state plasma levels thereafter were predicted to be higher than the EC for all genotypes tested ₉₅At least 2 times higher and 3 to 5 times more potent than GT2. This data indicates that the compound 2It has potent pan-genotypic antiviral activity in humans. As shown in Figure 24, the EC of sofosbuvir at GT1, GT3 and GT4 ₉₅greater than 100 ng/mL. Therefore, surprisingly, the compound 2Active against HCV at dosage forms that deliver lower steady-state minimum concentrations (40-50 ng/mL) than those achieved by similar sofosbuvir dosage forms (approximately 100 ng/mL). Examples 26 . Compound 2 Preparation Description and ManufacturingCompounds 2Representative non-limiting batch formulas for tablets (50 mg and 100 mg) are presented in Table 36. Tablets were produced by co-blending using a direct compression method, as shown in Figure 25. The active pharmaceutical ingredient (API) was adjusted based on the current analysis, with adjustments made in the percentage of microcrystalline cellulose. The API and excipients (microcrystalline cellulose, lactose monohydrate, and sodium croscarmellose) were screened, placed in a V blender (PK Blendmaster, 0.5 L tank) and mixed at 25 rpm for 5 minutes. Magnesium stearate was then screened, added, and the blend was mixed for an additional 2 minutes. The co-blend was distributed to produce 50 mg and 100 mg tablets. The lubricated blend was then compressed at 10 tablets/min using a single punch research tablet maker (Korsch XP1) and a gravimetric powder feeder. A round standard concave 6 mm tool and a force of 3.5 kN produced 50 mg tablets. An 8 mm round standard concave tool and a force of 3.9-4.2 kN produced 100 mg tablets. surface 36 . 50 mg and 100 mg Compound 2 Tablet preparation raw material % w/w g/ batch Mg/ unit 50 mg tablet 100 mg tablet Compound 2 50.0 180.0 50.0 100.0 Microcrystalline cellulose, USP/NF, EP 20.0 72.0 20.0 40.0 Lactose monohydrate, USP/NF, BP, EP, JP 24.0 86.4 24.0 48.0 Cross-linked carboxymethyl cellulose sodium, USP/NF, EP 5.0 18.0 5.0 10.0 Magnesium stearate, USP/NF, BP, EP, JP 1.0 3.6 1.0 2.0 Total 100.0 200.0 Adjust compounds based on current analysis 2, with adjustments made in the percentage of microcrystalline cellulose. Screening compounds 2and excipients (microcrystalline cellulose, lactose monohydrate and cross-linked carboxymethyl cellulose sodium), were placed in a V blender (PK Blendmaster, 0.5 L tank) and mixed at 25 rpm for 5 minutes. The blend was then screened, magnesium stearate was added and mixed for an additional 2 minutes. The co-blend was dispensed to produce 50 mg and 100 mg tablets. The blend was then compressed and lubricated using a single-shot research tablet maker (Korsch XP1) and a gravity powder feeder at a rate of 10 tablets/min. A round standard concave 6 mm tool and a force of 3.5 kN were used to produce 50 mg tablets. 100 mg tablets were produced using an 8 mm round standard concave tool and a force of 3.9-4.2 kN. Specifications for 50 mg and 100 mg tablets are shown in Table 37. surface 37 . Compound 2 Of 50 mg and 100 mg Tablet specifications 50 mg tablet 100 mg tablet Average weight (n=10) 100 ± 5 mg 200 ± 10 mg Individual weight 100 ± 10 mg 200 ± 20 mg hardness 5.3 kp 8.3 kp Split ＜ 15 minutes ＜ 15 minutes Crispness NMT 0.5% NMT 0.5% The 50 mg and 100 mg tablets produced as described above were subjected to a 6 month stability study at the following three conditions: 5°C (refrigerated), 25°C/60% RH (ambient), and 40°C/75% RH (accelerated). Both the 50 mg and 100 mg tablets were chemically stable at all three conditions tested. Under refrigerated conditions (5°C), both the 50 mg and 100 mg tablets remained morphologically unchanged white solids from T=0 months to T=6 months. No impurities greater than 0.05% were reported for either the 50 mg tablet or the 100 mg tablet throughout the 6 month study. The water content after 6 months was also less than 3.0% w/w for both tablets. Similar results were reported when the tablets were subjected to ambient conditions (25°C/60% RH), with no impurities greater than 0.05% reported for both tablets throughout the 6 months and the water content not exceeding 3.0% w/w at the 6 month mark. When the tablets were subjected to accelerated conditions (40°C/75% RH), the morphology of the 50 mg and 100 mg tablets did not change from white round tablets. One impurity was reported after 3 months, but the impurity was only 0.09%. A second impurity was reported after 6 months, but the total impurity percentage was only 0.21% for both the 50 mg and 100 mg tablets. The water content at 6 months was 3.4% w/w for the 50 mg tablet and 3.2% w/w for the 100 mg tablet. In an independent study, the compound was measured at ambient conditions (25°C/60% RH) 2The stability of 50 mg and 100 mg tablets over 9 months. The morphology of the 50 mg and 100 mg tablets did not change from white round tablets over the course of 9 months. The impurities in the 50 mg tablets were less than 0.10% after 9 months and the impurities in the 100 mg tablets were less than 0.05%. The water content of the 50 mg tablets and 100 mg tablets was only 2.7% w/w and 2.6% w/w, respectively, after 9 months. This specification has been described with reference to the embodiments of the present invention. However, it is generally understood by those skilled in the art that various modifications and variations may be made without departing from the scope of the invention as described in the patent application below. Therefore, this specification should be viewed in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.

FIG. 1A is an overlay of XRPD diffraction patterns of samples 1-1 (amorphous Compound 1), 1-2 (crystalline Compound 1 ), and 1-3 (amorphous Compound 2 ) prior to stability studies for characterization purposes, as described in Examples 2 and 5. The x-axis is 2θ measured in degrees, and the y-axis is intensity measured in counts. FIG. 1B is an HPLC analysis of amorphous Compound 1 (sample 1-1) to determine the purity as described in Example 2. The purity of the sample was 98.7%. The x-axis is time measured in minutes, and the y-axis is intensity measured in counts. FIG. 2A is an HPLC analysis of crystalline Compound 1 (sample 1-2) to determine the purity as described in Example 2. The purity of the sample was 99.11%. The x-axis is time measured in minutes, and the y-axis is intensity measured in counts. FIG. 2B is a DSC and TGA graph of crystalline Compound 1 (sample 1-2) prior to any stability studies for any characterization purposes, as described in Example 2. The x-axis is temperature measured in °C, the left y-axis is heat flow measured in (W/g), and the right y-axis is weight measured in percentage. FIG. 3 is an X-ray crystallographic image of Compound 1 showing absolute stereochemistry, as described in Example 2. FIG. 4A is an overlay of XRPD diffraction patterns of samples 1-1 (amorphous Compound 1 ), 1-2 (crystalline Compound 1 ), and 1-3 (amorphous Compound 2 ) after storage at 25°C and 60% relative humidity for 14 days, as described in Example 2. The x-axis is 2θ measured in meters, and the y-axis is intensity measured in counts. FIG4B is an overlay of XRPD diffraction patterns of samples 1-4, 1-5, 1-6, 1-7, and 1-9 after storage for 7 days at 25° C. and 60% relative humidity, as described in Example 4. The x-axis is 2θ measured in meters, and the y-axis is intensity measured in counts. FIG5A is an overlay of XRPD diffraction patterns of samples 1-4, 1-6, 1-7, and 1-9 after storage for 14 days at 25° C. and 60% relative humidity, as described in Example 4. The x-axis is 2θ measured in meters, and the y-axis is intensity measured in counts. FIG. 5B is an XRPD graph of amorphous Compound 2 (Samples 1-3), as described in Example 5. The x-axis is 2θ measured in degrees, and the y-axis is intensity measured in counts. FIG. 6A is an HPLC analysis of amorphous Compound 2 (Samples 1-3) to determine purity, as described in Example 5. The purity of the sample was 99.6%. The x-axis is time measured in minutes, and the y-axis is intensity measured in counts. FIG. 6B is a DSC and TGA graph of amorphous Compound 2 (Samples 1-3) prior to any stability studies for characterization purposes, as described in Example 5. The x-axis is temperature measured in ° C, the left y-axis is heat flow measured in (W/g), and the right y-axis is weight measured in percentage. FIG. 7A is an overlay of XRPD diffraction patterns of crystalline samples (samples 2-2, 2-6, and 2-7) and poorly crystalline samples (samples 2-3, 2-4, 2-5, and 2-8) identified from the crystallization of Compound 2 (Example 6). The x-axis is 2θ measured in metric terms, and the y-axis is the intensity measured in counts. FIG. 7B is an overlay of XRPD diffraction patterns of amorphous samples (samples 2-9, 2-10, and 2-11) identified from the crystallization of Compound 2 (Example 6). The x-axis is 2θ measured in metric terms, and the y-axis is the intensity measured in counts. FIG8A is an overlay of XRPD diffraction patterns of samples (samples 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, and 2-8) after storage at 25° C. and 60% relative humidity for 6 days (Example 6). The x-axis is 2θ measured in degrees, and the y-axis is intensity measured in counts. FIG8B is a DSC and TGA graph of sample 2-2 (Example 6). The x-axis is temperature measured in degrees Celsius, the left y-axis is heat flow measured in (W/g), and the right y-axis is weight measured in percentage. The experimental procedure for DSC and TGA acquisition is given in Example 2. FIG9A is a DSC and TGA graph of sample 2-3 (Example 6). The x-axis is the temperature measured in °C, the left y-axis is the heat flow measured in (W/g), and the right y-axis is the weight measured in percentage. The experimental procedure for DSC and TGA acquisition is given in Example 2. Figure 9B is a DSC and TGA graph of sample 2-4 (Example 6). The x-axis is the temperature measured in °C, the left y-axis is the heat flow measured in (W/g), and the right y-axis is the weight measured in percentage. The experimental procedure for DSC and TGA acquisition is given in Example 2. Figure 10A is a DSC and TGA graph of sample 2-5 (Example 6). The x-axis is the temperature measured in °C, the left y-axis is the heat flow measured in (W/g), and the right y-axis is the weight measured in percentage. The experimental procedure for DSC and TGA collection is given in Example 2. Figure 10B is a DSC and TGA graph of sample 2-6 (Example 6). The x-axis is the temperature measured in ° C, the left y-axis is the heat flow measured in (W/g), and the right y-axis is the weight measured in percentage. The experimental procedure for DSC and TGA collection is given in Example 2. Figure 11A is a DSC and TGA graph of sample 2-7 (Example 6). The x-axis is the temperature measured in ° C, the left y-axis is the heat flow measured in (W/g), and the right y-axis is the weight measured in percentage. The experimental procedure for DSC and TGA collection is given in Example 2. Figure 11B is a DSC and TGA graph of sample 2-8 (Example 6). The x-axis is temperature measured in °C, the left y-axis is heat flow measured in (W/g), and the right y-axis is weight measured in percentage. The experimental procedure for DSC and TGA acquisition is given in Example 2. Figure 12A is an XRPD pattern of amorphous Compound 4 (sample 3-12) as discussed in Example 7. The x-axis is 2θ measured in degrees, and the y-axis is intensity measured in counts. Regardless of the solvent used, no crystallization of the malonate salt was observed. Figure 12B is an overlay of the XRPD diffraction patterns of amorphous samples (samples 3-6, 3-10, 3-11 and 3-12) identified from attempts to crystallize Compound 1 with malonate salt (Example 7). The x-axis is 2θ measured in metric and the y-axis is intensity measured in counts. FIG. 13A is an HPLC chromatogram of sample 3-12 from an attempt to crystallize Compound 1 with the malonate salt, as described in Example 7. The sample was 99.2% pure. The x-axis is time measured in minutes and the y-axis is intensity measured in mAu. FIG. 13B is an overlay of XRPD diffraction patterns of solid samples (samples 4-13, 4-12, 4-9, 4-3, and 4-1) obtained from crystallization using LAG compared to Compound 1 (sample 1-2), as described in Example 8. All XRDPs match the pattern of the crystallized acid counterion with no additional peaks. The x-axis is 2θ measured in metric and the y-axis is intensity measured in counts. FIG. 14A is an overlay of XRPD diffraction patterns of samples (samples 6-13, 6-12, 6-11, 6-10, 6-8, 6-7, 6-6, 6-5, 6-4, and 6-2) obtained from using ethyl acetate as a crystallization solvent compared to crystallized Compound 1 (sample 1-2), as described in Example 10. The XRPD pattern was found to generally match that of Compound 1 , except for samples 6-2, 6-4, and 6-5, which exhibited slight differences. The x-axis is 2θ measured in degrees, and the y-axis is intensity measured in counts. FIG. 14B is an overlay of XRPD diffraction patterns of sample 5-1 after a second dissolution in MEK and the addition of antisolvents cyclohexane and penic acid, as described in Example 9. Sample 5-1 crystallized in palmitic acid was solid after synthesis, but the XRPD pattern matched the palmitic acid pattern. FIG. 15A is an overlay of XRPD diffraction patterns of samples (samples 6-5, 6-4, and 6-2) obtained from using ethyl acetate as a crystallization solvent compared to crystallized compound 1 (sample 1-2), as described in Example 10. It was found that the XRPD pattern generally matched the compound 1 pattern except for samples 6-2, 6-4, and 6-5, which showed slight differences. The x-axis is 2θ measured in metric units, and the y-axis is the intensity measured in counts and labeled with the acid used in the crystallization. FIG. 15B is an XRPD pattern of compound 2 , as described in Example 14. The x-axis is 2θ measured in metric units, and the y-axis is the intensity measured in counts. Figure 16A is a graph of active TP (metabolites 1-6 ) concentration levels in the liver and heart of rats, dogs, and monkeys (Example 18). The x-axis is the dose measured in mg/kg for each species, and the y -axis is the active TP concentration measured in ng/ g . Figure 16B is a graph of active TP (metabolites 1-6 ) concentration levels in the liver and heart of dogs (n = 2) measured 4 hours after a single oral dose of Compound 1 or Compound 2 (Example 19). The x-axis is the dose of each compound measured in mg/kg, and the y-axis is the active TP concentration measured in ng/g. Figure 17 is a plasma curve of compound 1 and metabolites 1-7 measured 72 hours after administration in rats given a single 500 mg/kg oral dose of compound 2 (Example 20). The x-axis is time measured in hours, and the y-axis is plasma concentration measured in ng/mL. Figure 18 is a plasma curve of compound 1 and metabolites 1-7 measured 72 hours after administration in monkeys given a single oral dose of 30 mg, 100 mg or 300 mg of compound 2 (Example 20 ). The x-axis is time measured in hours, and the y-axis is plasma concentration measured in ng/mL. FIG. 19 is a graph of the EC ₉₅ of Sofosbuvir and Compound 1 against HCV clinical isolates measured in nM. The EC ₉₅ values of Compound 1 are 7 to 33 times lower than those of Sofosbuvir (Example 22). The x-axis is labeled with genotype, and the y-axis is the EC ₉₅ measured in nM. FIG. 20 is a graph of the EC 50 of Sofosbuvir and Compound 1 against laboratory strains of HCV genotypes 1a, 1b, 2a, 3a, 4a, and 5a measured in nM. In genotypes 1-5, Compound 1 is approximately 6 to 11 times more potent than Sofosbuvir (Example 22). The x-axis is labeled with genotype, and the y-axis is _{the EC 50 measured} in nM. Figure 21 is a graph of the mean plasma concentration-time curves of Compound 1 after a single dose of Compound 2 was administered to all groups in Part B of the study, as described in Example 24. Compound 1 is rapidly absorbed and rapidly metabolized in approximately 8 hours in all groups from Part B. The x-axis is time measured in hours, and the y-axis is the geometric mean plasma concentration measured in ng/mL. Figure 22 is a graph of the mean plasma concentration- time curves of metabolites 1-7 after a single dose of Compound 2 was administered to all groups in Part B of the study , as described in Example 24. Metabolites 1-7 presented stable plasma concentrations in all groups from Part B. The x-axis is time measured in hours, and the y-axis is the geometric mean plasma concentration measured in ng/mL. Figure 23A is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the Participant 1b population, as described in Example 24. The graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dotted line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value for GT1b . The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng/mL, and the right y -axis is the HCV RNA reduction measured in log ₁₀ IU / mL. FIG. 23B is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the 1b cohort, as described in Example 24. The graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dashed line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value for GT1b . The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng/mL, and the right y-axis is the HCV RNA reduction measured in log ₁₀ IU / mL. FIG. 23C is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the 1b cohort, as described in Example 24. The graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dotted line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value for GT1b. The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng /mL, and the right y-axis is the HCV RNA reduction measured in log ₁₀ IU/mL. Figure 23D is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the participant 3b population, as described in Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dotted line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value for GT1b. The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng/mL, and the right y-axis is the reduction in HCV RNA measured in log ₁₀ IU / mL. Figure 23E is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the participant 3b population, as described in Example 24. Each figure shows plasma metabolite 1-7 exposure and HCV RNA reduction levels . The dotted line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value for GT1b . The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng/mL, and the right y-axis is the reduction of HCV RNA measured in log ₁₀ IU / mL. FIG . 23F is an individual pharmacokinetic/pharmacodynamic analysis of individuals in the participant 3b population, as described in Example 24. Each graph shows plasma metabolite 1-7 exposure and HCV RNA reduction levels. The dotted line represents the minimum concentration of metabolites 1-7 required to maintain a viral response greater than the EC ₉₅ value against GT1b . The x-axis is time measured in hours. The left y-axis is the plasma concentration of metabolites 1-7 measured in ng/mL, and the right y-axis is the reduction in HCV RNA measured in log ₁₀ IU / mL. Figure 24 is a graph of the EC ₉₅ values of compound 1 and sofosbuvir against clinical isolates of patients with GT1, GT2, GT3, and GT4 HCV infection. The horizontal dashed line (- - - - -) represents the steady-state minimum concentration of sofosbuvir nucleoside (C _{24 , ss} ) after a dose of 400 mg QD sofosbuvir. The full horizontal line ( ) represents the steady-state minimum concentration (C _{24 , ss} ) of metabolites 1 - 7 after 600 mg of compound 2 (equivalent to 550 mg of compound 1 ). The horizontal dotted line (---------) represents the steady-state minimum concentration (C _{24 , ss} ) of metabolites 1 - 7 after 450 mg of compound 2 (equivalent to 400 mg of compound 1 ). As discussed in Example 25, the predicted steady-state minimum plasma levels (C _{24 , ss} ) of metabolites 1 - 7 after 600 mg and 450 mg of compound 2 exceeded the in vitro EC ₉₅ of compound 1 against all clinical isolates tested. The steady-state minimum plasma level of sofosbuvir (C _{24 , ss} ) just exceeded the EC ₉₅ under the GT2 clinical isolate. The x-axis is labeled with the clinical isolate, and the table below the x-axis lists the EC ₉₅ values of compound 1 and sofosbuvir. The y-axis is the EC ₉₅ for the clinical isolate measured in ng/mL. EC ₉₅ is expressed as nucleoside equivalents. Sofosbuvir and compound 2 were administered daily (QD). Figure 25 is a flow chart showing the manufacturing process of compound 2 for 50 mg and 100 mg tablets, as described in Example 26. In step 1, microcrystalline cellulose, compound 2 , lactose monohydrate, and cross-linked carboxymethyl cellulose sodium were filtered through a 600 μM sieve. In step 2, the contents from step 1 were loaded into a V-blender and mixed at 25 rpm for 5 minutes. In step 3, magnesium stearate was filtered through a 600 μM sieve. In step 4, magnesium stearate was loaded into a V-blender containing the contents from step 2 (microcrystalline cellulose, compound 2 , lactose monohydrate, and cross-linked carboxymethyl cellulose sodium) and mixed at 25 rpm for 2 minutes. The co-blend was then distributed to produce 50 mg tablets and 100 mg tablets. To produce 50 mg tablets, the blend from step 4 was compressed using a 6 mm round standard concave tool. To produce 100 mg tablets, the blend from step 4 was compressed using an 8 mm round standard concave tool. The tablets were then packaged into HDPE bottles induction sealed with dry PP caps. Figure 26 shows that the hemisulfate salt exhibits favorable pharmacological properties over its corresponding free base for the treatment of HCV virus.

Claims (36)

A compound of the formula: . The compound of claim 1 has the following formula: . The compound of claim 1 has the following formula: . The compound of claim 1 has the following formula: . The compound of claim 1 has the following formula: . A compound of the formula: . The compound of claim 6, wherein the compound is at least 90% free of the opposite phosphorus S enantiomer. The compound of claim 6, wherein the compound is at least 98% free of the opposite phosphorus S enantiomer. The compound of claim 6, wherein the compound is at least 99% free of the opposite phosphorus S enantiomer. The compound of claim 5 has the following formula: . The compound of claim 10, wherein the compound is at least 90% free of the opposite phosphorus R enantiomer. The compound of claim 5 has the following formula: . The compound of claim 12, wherein the compound is at least 90% free of the opposite phosphorus S enantiomer. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 13 in a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 14 is in the form of an oral dosage form. A pharmaceutical composition as claimed in claim 15, wherein the oral dosage form is a solid dosage form. The pharmaceutical composition of claim 16, wherein the solid dosage form is a tablet. The pharmaceutical composition of claim 16, wherein the solid dosage form is a capsule. A pharmaceutical composition as claimed in claim 15, wherein the oral dosage form is a liquid dosage form. The pharmaceutical composition of claim 19, wherein the liquid dosage form is a suspension or a solution. The pharmaceutical composition of claim 14, which is in the form of an intravenous formulation. The pharmaceutical composition of claim 14, which is in the form of a parenteral formulation. The pharmaceutical composition of claim 14, which delivers at least 600 mg of the compound. A use of a therapeutically effective amount of a compound as claimed in any one of claims 1 to 13, optionally in a pharmaceutically acceptable carrier, for the preparation of a medicament for the treatment of HCV in humans. The use as claimed in claim 24, wherein the drug is administered orally. The use as claimed in claim 24, wherein the drug is administered intravenously. The use as claimed in claim 24, wherein the drug is administered parenterally. The use of claim 25, wherein at least 600 mg of the compound is used. The use as claimed in claim 28, wherein the drug is taken once a day. The use as claimed in claim 28, wherein the drug is taken twice a day. The use as claimed in claim 28, wherein the drug is taken three times a day. The use of claim 25, wherein the hepatitis C virus is of genotype 1a or 1b. The use of claim 25, wherein the hepatitis C virus is of genotype 2a or 2b. The use of claim 25, wherein the hepatitis C virus is genotype 3a. The use of claim 25, wherein the hepatitis C virus is of genotype 4a or 4d. The use of claim 25, wherein the hepatitis C virus is of genotype 5a.